######## snakemake preamble start (automatically inserted, do not edit) ########
import sys;sys.path.extend(['/fh/fast/bloom_j/software/miniforge3/envs/seqneut-pipeline/lib/python3.13/site-packages', '/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025/seqneut-pipeline', '/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025', '/fh/fast/bloom_j/software/miniforge3/envs/seqneut-pipeline/bin', '/fh/fast/bloom_j/software/miniforge3/envs/seqneut-pipeline/lib/python3.13', '/fh/fast/bloom_j/software/miniforge3/envs/seqneut-pipeline/lib/python3.13/lib-dynload', '/fh/fast/bloom_j/software/miniforge3/envs/seqneut-pipeline/lib/python3.13/site-packages', '/home/jbloom/.cache/snakemake/snakemake/source-cache/runtime-cache/tmpfro_n8p6/file/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025/seqneut-pipeline/notebooks', '/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025/seqneut-pipeline/notebooks']);import pickle;from snakemake import script;script.snakemake = 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\x8c\x10ACACGGGTTGGCTGTA\x94K!\x8c\x10ACAGTACGATCTACGC\x94K"\x8c\x10ACAGTCCACCATTGAG\x94K#\x8c\x10ACATTTTCCAATAGGT\x94K$\x8c\x10ACCAGCAATGAGTTGT\x94K%\x8c\x10ACCCCCGGAGCTTGGC\x94K&\x8c\x10ACCGAATGAATCATCC\x94K\'\x8c\x10ACCGATTCACGAATAA\x94K(\x8c\x10ACCGTTGTACACACCA\x94K)\x8c\x10ACGCAAATAGACCGAA\x94K*\x8c\x10ACGGGGATTGGCTGTT\x94K+\x8c\x10ACGTATGATTTTCGAG\x94K,\x8c\x10ACGTCCATTAAGATCA\x94K-\x8c\x10ACGTGTCTCCGAGCAA\x94K.\x8c\x10ACTACGAGGCTACGTA\x94K/\x8c\x10ACTCTGGCTCGCTAAT\x94K0\x8c\x10ACTGTCTAGAAATTTT\x94K1\x8c\x10AGAAAATCTCAGATAC\x94K2\x8c\x10AGACCATCGCACCCAA\x94K3\x8c\x10AGACCGCCAGTTTCGT\x94K4\x8c\x10AGAGCTAAAAAGAGGA\x94K5\x8c\x10AGATCCACCCTATAGT\x94K6\x8c\x10AGATCCCAGGTCCTTT\x94K7\x8c\x10AGCATAGGGATATGTG\x94K8\x8c\x10AGCATCTAACAGATAG\x94K9\x8c\x10AGCCCATGCTGGGGAT\x94K:\x8c\x10AGCGACATCGCCCTTT\x94K;\x8c\x10AGCTCCTGGGGTATCA\x94K<\x8c\x10AGCTGAATTAAGTATG\x94K=\x8c\x10AGGAAAGAAACTGGAG\x94K>\x8c\x10AGGACTATAGTTGGCA\x94K?\x8c\x10AGGAGTATGAAGAGCG\x94K@\x8c\x10AGGCCCGTAAGGACTA\x94KA\x8c\x10AGGTTCAGACTCTTGC\x94KB\x8c\x10AGTAAACATGCATTGG\x94KC\x8c\x10AGTATTTGCGCTTCAA\x94KD\x8c\x10AGTCCTATCCTCAAAT\x94KE\x8c\x10AGTCGTTTAGATAGTT\x94KF\x8c\x10AGTGTTGAATAGGCGA\x94KG\x8c\x10AGTGTTGGCTTGGTTA\x94KH\x8c\x10AGTTCCATAGGCATGG\x94KI\x8c\x10AGTTGGGGTCTCCCTT\x94KJ\x8c\x10AGTTTTTATAACTTGC\x94KK\x8c\x10ATAACGTTTGTGCAAA\x94KL\x8c\x10ATAACTGAGGGCATTG\x94KM\x8c\x10ATACACGAGGTTGTGA\x94KN\x8c\x10ATACACGCATGTGCCA\x94KO\x8c\x10ATAGAAAATTATCCGC\x94KP\x8c\x10ATAGAATCGCAAATTA\x94KQ\x8c\x10ATAGGATATATGGCTG\x94KR\x8c\x10ATATAAAAAACTTAGT\x94KS\x8c\x10ATCAGGATAATCGCGC\x94KT\x8c\x10ATCCGATTTAAAGGCA\x94KU\x8c\x10ATGGCCCACGGGCATA\x94KV\x8c\x10ATGGGATTGGAGAAAC\x94KW\x8c\x10ATGGTTTTACGTCCAT\x94KX\x8c\x10ATTAGATTATAACGTA\x94KY\x8c\x10ATTAGGGCTACGTGAG\x94KZ\x8c\x10ATTATCATATCTAATA\x94K[\x8c\x10ATTCCGAATGGGGTAG\x94K\\\x8c\x10ATTTAAATTCGAGGAC\x94K]\x8c\x10ATTTACTCATTATACG\x94K^\x8c\x10ATTTTTCTATGGCTAC\x94K_\x8c\x10CAAAAGCAGCACGATA\x94K`\x8c\x10CAAAATCTACGGCGAC\x94Ka\x8c\x10CAAATGCTGCATTAGG\x94Kb\x8c\x10CAATTCGCCGTTCCCC\x94Kc\x8c\x10CACAGACAATAAAAAA\x94Kd\x8c\x10CACCAATCTTCGAACT\x94Ke\x8c\x10CACCATCAGCACCTAG\x94Kf\x8c\x10CACCGCGCCGAGCACC\x94Kg\x8c\x10CACCTAGGATCGCACT\x94Kh\x8c\x10CACGGCCGGCGAACTC\x94Ki\x8c\x10CACGGGCTAATGTCTC\x94Kj\x8c\x10CACTAGATGTACAGTC\x94Kk\x8c\x10CAGAACCTCGTTGTCT\x94Kl\x8c\x10CAGATAGTGATGAACA\x94Km\x8c\x10CAGGCTCTAGAGCTCT\x94Kn\x8c\x10CATAAAAGACTGTATA\x94Ko\x8c\x10CATGGGAATTGCCACT\x94Kp\x8c\x10CATGTGGAGCCCAACA\x94Kq\x8c\x10CATTGAGACGCGCAAG\x94Kr\x8c\x10CATTTCTGATGAATTG\x94Ks\x8c\x10CCAACACAAAAAATTA\x94Kt\x8c\x10CCAAGCTTGGCGCATC\x94Ku\x8c\x10CCCCCGCTGTTTAAAA\x94Kv\x8c\x10CCCCTCCTCTAAAGTT\x94Kw\x8c\x10CCCTATGAAATAAGCT\x94Kx\x8c\x10CCCTGCGCGGCTCGGG\x94Ky\x8c\x10CCCTTTACGGATCTCT\x94Kz\x8c\x10CCGCATTAGCGGGAGG\x94K{\x8c\x10CCGCGCACGTTTAGAG\x94K|\x8c\x10CCGGATAAATCAGAAC\x94K}\x8c\x10CCTGGGTTAAGTTGTG\x94K~\x8c\x10CCTTGATGCATTCCCG\x94K\x7f\x8c\x10CCTTTCTCAAAACATA\x94K\x80\x8c\x10CGAAAACATTACAAAT\x94K\x81\x8c\x10CGAAACACGTCCCAGT\x94K\x82\x8c\x10CGAACCGCAGACACGT\x94K\x83\x8c\x10CGACTCCACGGACGCC\x94K\x84\x8c\x10CGATCTTTACGAAAAA\x94K\x85\x8c\x10CGCACTTTACGAGACA\x94K\x86\x8c\x10CGCAGCATTGGTCGCC\x94K\x87\x8c\x10CGCGACACCCTTCCGG\x94K\x88\x8c\x10CGGACCCTAGATGGTA\x94K\x89\x8c\x10CGGCCAGGGAATCAAA\x94K\x8a\x8c\x10CGGGAAATGTAAATGA\x94K\x8b\x8c\x10CGGGAACATACATAAC\x94K\x8c\x8c\x10CGGGAATCTCCCATAC\x94K\x8d\x8c\x10CGGGGACAAGATTGTA\x94K\x8e\x8c\x10CGGTCGGGACTCATCT\x94K\x8f\x8c\x10CGTACAGTGTAATCGA\x94K\x90\x8c\x10CGTACGTATGTCCCAG\x94K\x91\x8c\x10CGTGACCCCCTCCAAC\x94K\x92\x8c\x10CGTGTACCCTTCAGCA\x94K\x93\x8c\x10CGTTAACGGCCTATCC\x94K\x94\x8c\x10CGTTCAGCGATAACGG\x94K\x95\x8c\x10CGTTTTTGGTTCGAGG\x94K\x96\x8c\x10CTAATTTAAGTATCAA\x94K\x97\x8c\x10CTAGCACAGCGTAGGC\x94K\x98\x8c\x10CTATAAACCGTTTGTA\x94K\x99\x8c\x10CTATATTGCCCGGAAG\x94K\x9a\x8c\x10CTATCTTAATCTACAG\x94K\x9b\x8c\x10CTATTTAACAGACGTA\x94K\x9c\x8c\x10CTCAAATAATTGGCGC\x94K\x9d\x8c\x10CTCAATGTCGTAGGAT\x94K\x9e\x8c\x10CTCCTAGGGGACGATT\x94K\x9f\x8c\x10CTCTTACGCTCCTACG\x94K\xa0\x8c\x10CTGAACTTATCTGTGG\x94K\xa1\x8c\x10CTGAACTTGTCGATAT\x94K\xa2\x8c\x10CTGAGCTGCCAATAAG\x94K\xa3\x8c\x10CTGAGGGATTCAACTC\x94K\xa4\x8c\x10CTGGAGGCCTGGCCCC\x94K\xa5\x8c\x10CTGTACCTGCAGTTGA\x94K\xa6\x8c\x10CTTACAGAATACTAGA\x94K\xa7\x8c\x10CTTACTGCGCGAGAGT\x94K\xa8\x8c\x10CTTAGGTATTATATGC\x94K\xa9\x8c\x10CTTCGGCTCTTGATTA\x94K\xaa\x8c\x10CTTTTCTAGTACGCTT\x94K\xab\x8c\x10GAAAGCCCCGTGCAAT\x94K\xac\x8c\x10GAAAGTCCCTATGATG\x94K\xad\x8c\x10GAAATCCCCAAATAAC\x94K\xae\x8c\x10GAAGAAACTATAACCA\x94K\xaf\x8c\x10GAAGTACGCTGAATGA\x94K\xb0\x8c\x10GAAGTGCGTATTGAGT\x94K\xb1\x8c\x10GAAGTGCTGCTGAAGT\x94K\xb2\x8c\x10GAATAATAGAACAGAG\x94K\xb3\x8c\x10GACAAAAGGGACATAT\x94K\xb4\x8c\x10GACCCCTTGTAAGATG\x94K\xb5\x8c\x10GACGGGATGGGCACGT\x94K\xb6\x8c\x10GAGAGCTGCAGAAGCG\x94K\xb7\x8c\x10GAGCTTGCTATGGATC\x94K\xb8\x8c\x10GAGGGGATACGTCACC\x94K\xb9\x8c\x10GAGGGGTAGAGATACG\x94K\xba\x8c\x10GATCACGCAGAAAAAG\x94K\xbb\x8c\x10GATCGCCACTGATAAG\x94K\xbc\x8c\x10GATCGCCATCGACTTC\x94K\xbd\x8c\x10GATCTTGGGGAGAGTC\x94K\xbe\x8c\x10GATTCAGATGCCCACC\x94K\xbf\x8c\x10GCAAACAGTGTAGTTG\x94K\xc0\x8c\x10GCAACGAGGTGTAACC\x94K\xc1\x8c\x10GCAGCGTGCCGGTCAT\x94K\xc2\x8c\x10GCATCCTCAACTCCTA\x94K\xc3\x8c\x10GCATGGAACTAACTCC\x94K\xc4\x8c\x10GCCAGCTCGTATCCCT\x94K\xc5\x8c\x10GCCATTTACTGAAGGG\x94K\xc6\x8c\x10GCCGCTGCGGCGTGTG\x94K\xc7\x8c\x10GCCGGCGTTAGTGTCA\x94K\xc8\x8c\x10GCCTTTGCGCGCAGTC\x94K\xc9\x8c\x10GCGAAGTTTCATAGCG\x94K\xca\x8c\x10GGTTAACTTTGGAAGC\x94K\xcb\x8c\x10GTAAGCAAAGTTGACC\x94K\xcc\x8c\x10GTAAGCTTCATGGAGT\x94K\xcd\x8c\x10GTAATTCGCATGCGGA\x94K\xce\x8c\x10GTACCCAGTTCCTGCG\x94K\xcf\x8c\x10GTAGAACTGCGGCCCC\x94K\xd0\x8c\x10GTAGATACTAGGACCA\x94K\xd1\x8c\x10GTCAAGTTACGGATGG\x94K\xd2\x8c\x10GTCCGTCAGCATAAAC\x94K\xd3\x8c\x10GTCGCATCCTGGAATG\x94K\xd4\x8c\x10GTCGCCGCTAATCCGA\x94K\xd5\x8c\x10GTGAGCGAGAAAAGCA\x94K\xd6\x8c\x10GTGCATCCTAGTGACG\x94K\xd7\x8c\x10GTGCGATTGTCCGGAA\x94K\xd8\x8c\x10GTGGTATCAAGCCGGG\x94K\xd9\x8c\x10GTTATTATGACTTCAT\x94K\xda\x8c\x10GTTGCTCCGACACGCC\x94K\xdb\x8c\x10TAAAAAGCCTCCATGA\x94K\xdc\x8c\x10TAACGTGATTTCTCGA\x94K\xdd\x8c\x10TAATAAGCCAGCAAGA\x94K\xde\x8c\x10TACAAGAGAGGGGTCC\x94K\xdf\x8c\x10TACATACCGACGCAGT\x94K\xe0\x8c\x10TACCAATGTCATTTGA\x94K\xe1\x8c\x10TACCTGCTGCGGAACG\x94K\xe2\x8c\x10TACTAATGCCGTTGTC\x94K\xe3\x8c\x10TACTAGCAATAAAATC\x94K\xe4\x8c\x10TACTGATAACCCTGCG\x94K\xe5\x8c\x10TAGCATTGTCGGAAAG\x94K\xe6\x8c\x10TAGCTGATAGTAACTC\x94K\xe7\x8c\x10TATATTAGTAACATAA\x94K\xe8\x8c\x10TATCCAAGGGACGGAC\x94K\xe9\x8c\x10TATGTCGTATCCACAG\x94K\xea\x8c\x10TATTAAGAGAAGTGCG\x94K\xeb\x8c\x10TATTCCTAACTAGCGA\x94K\xec\x8c\x10TCAATCGGGGGCTAAA\x94K\xed\x8c\x10TCACGACTCGACTAAC\x94K\xee\x8c\x10TCATGGGTGTACGAGA\x94K\xef\x8c\x10TCCACACCCCTAGCTA\x94K\xf0\x8c\x10TCCAGCGCGGTAAGAG\x94K\xf1\x8c\x10TCCCCGTGGTTTGACA\x94K\xf2\x8c\x10TCGAACGAAGTAGGAG\x94K\xf3\x8c\x10TCGAGTTAATATGCGC\x94K\xf4\x8c\x10TCGATTACTAGCCGGA\x94K\xf5\x8c\x10TCGCTTCAACTAAAAA\x94K\xf6\x8c\x10TCGTCCGTTGGGAACT\x94K\xf7\x8c\x10TCGTCGCACTACTGCT\x94K\xf8\x8c\x10TCTAACTCTCGCGGCA\x94K\xf9\x8c\x10TCTCAGCTCTTAGCCG\x94K\xfa\x8c\x10TCTGGAAACGATCCCC\x94K\xfb\x8c\x10TCTTAGAGTGAACGAT\x94K\xfc\x8c\x10TCTTAGTCCTCGTATG\x94K\xfd\x8c\x10TCTTATTAGGCGGCAT\x94K\xfe\x8c\x10TCTTGACATAGCGATG\x94K\xff\x8c\x10TCTTTACCACTGCATC\x94M\x00\x01\x8c\x10TGACAACAATACAAAT\x94M\x01\x01\x8c\x10TGAGTTCATAGCTCCA\x94M\x02\x01\x8c\x10TGATCTGTGACATTGC\x94M\x03\x01\x8c\x10TGATCTTTTACATTTA\x94M\x04\x01\x8c\x10TGCAGTGGTATACATA\x94M\x05\x01\x8c\x10TGCGGTGGTCGATCCG\x94M\x06\x01\x8c\x10TGCTATTCCGGCGCGG\x94M\x07\x01\x8c\x10TGGAATCGTCACCGAT\x94M\x08\x01\x8c\x10TGGTCCGCTTCATGCT\x94M\t\x01\x8c\x10TGTAATAGGCGTCACA\x94M\n\x01\x8c\x10TGTCCGGATAAAGTAG\x94M\x0b\x01\x8c\x10TGTGGAGCGCCCTTAC\x94M\x0c\x01\x8c\x10TGTTGAGCCAGTCTGA\x94M\r\x01\x8c\x10TGTTGTAATCTGAATA\x94M\x0e\x01\x8c\x10TTAATGTAGCCGCTCC\x94M\x0f\x01\x8c\x10TTACGAATTTGATTCC\x94M\x10\x01\x8c\x10TTCATCAAGTTGGTGC\x94M\x11\x01\x8c\x10TTCTGTCCAGACTCGT\x94M\x12\x01\x8c\x10TTGAAAAAATCATAAA\x94M\x13\x01\x8c\x10TTGACTCACCGAATAA\x94M\x14\x01\x8c\x10TTGCAATTGAAACATA\x94M\x15\x01\x8c\x10TTGCTCCTGAGTAGTA\x94M\x16\x01\x8c\x10TTGGGCACTAAATTAA\x94M\x17\x01\x8c\x10TTGGGGAACGGGAAGC\x94M\x18\x01\x8c\x10TTGTATCAGTCGCGCC\x94M\x19\x01\x8c\x10TTTATATCCAACACCA\x94M\x1a\x01\x8c\x10TTTATATCGAGATTCA\x94M\x1b\x01\x8c\x10TTTCACAGAACCTATC\x94M\x1c\x01\x8c\x10TTTCAGCGTTGTTTTG\x94M\x1d\x01\x8c\x10TTTCGTGATACTCACA\x94u\x8c\x06strain\x94}\x94(K\x00\x8c\x19A/Minnesota/126/2024_H3N2\x94K\x01\x8c*A/Singapore/INFIMH-16-0019/2016X-307A_H3N2\x94K\x02\x8c\x18A/Wisconsin/67/2022_H1N1\x94K\x03\x8c\x16A/Lisboa/216/2023_H3N2\x94K\x04\x8c\x14A/Darwin/9/2021_H3N2\x94K\x05\x8c 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human serum samples collected in late 2024-2025 and combined pdmH1N1 and H3N2 influenza library\n\nThe numerical data and computer code are at 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script;from snakemake.logging import logger;from snakemake.script import snakemake;import os; os.chdir(r'/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025');
######## snakemake preamble end #########

Process plate counts to get fraction infectivities and fit curves

This notebook is designed to be run using snakemake, and analyzes a plate of sequencing-based neutralization assays.

The plots generated by this notebook are interactive, so you can mouseover points for details, use the mouse-scroll to zoom and pan, and use interactive dropdowns at the bottom of the plots.

Setup

Import Python modules:

import pickle
import sys
import warnings

import altair as alt

import matplotlib.pyplot as plt
import matplotlib.style

import neutcurve
from neutcurve.colorschemes import CBPALETTE, CBMARKERS

import numpy

import pandas as pd

import ruamel.yaml as yaml

_ = alt.data_transformers.disable_max_rows()

# avoid clutter w RuntimeWarning during curve fitting
warnings.filterwarnings("ignore", category=RuntimeWarning)

# faster plotting of neut curves
matplotlib.style.use("fast")

Get the variables passed by snakemake:

count_csvs = snakemake.input.count_csvs
fate_csvs = snakemake.input.fate_csvs
notebook_funcs = snakemake.input.notebook_funcs
qc_drops_yaml = snakemake.output.qc_drops
frac_infectivity_csv = snakemake.output.frac_infectivity_csv
fits_csv = snakemake.output.fits_csv
fits_pickle = snakemake.output.fits_pickle
viral_barcodes = snakemake.params.viral_barcodes
neut_standard_barcodes = snakemake.params.neut_standard_barcodes
samples = snakemake.params.samples
plate = snakemake.wildcards.plate
plate_params = snakemake.params.plate_params
curve_display_method = snakemake.params.curve_display_method

# get thresholds turning lists into tuples as needed
manual_drops = {
    filter_type: [tuple(w) if isinstance(w, list) else w for w in filter_drops]
    for (filter_type, filter_drops) in plate_params["manual_drops"].items()
}
group = plate_params["group"]
qc_thresholds = plate_params["qc_thresholds"]
curvefit_params = plate_params["curvefit_params"]
curvefit_qc = plate_params["curvefit_qc"]
curvefit_qc["barcode_serum_replicates_ignore_curvefit_qc"] = [
    tuple(w) for w in curvefit_qc["barcode_serum_replicates_ignore_curvefit_qc"]
]

print(f"Processing {plate=}")

samples_df = pd.DataFrame(plate_params["samples"])
print(f"\nPlate has {len(samples)} samples (wells)")
assert all(
    (len(samples_df) == samples_df[c].nunique())
    for c in ["well", "sample", "sample_noplate"]
)
assert len(samples_df) == len(
    samples_df.groupby(["serum_replicate", "dilution_factor"])
)
assert len(samples) == len(count_csvs) == len(fate_csvs) == len(samples_df)

for d, key, title in [
    (manual_drops, "manual_drops", "Data manually specified to drop:"),
    (qc_thresholds, "qc_thresholds", "QC thresholds applied to data:"),
    (curvefit_params, "curvefit_params", "Curve-fitting parameters:"),
    (curvefit_qc, "curvefit_qc", "Curve-fitting QC:"),
]:
    print(f"\n{title}")
    yaml.YAML(typ="rt").dump({key: d}, stream=sys.stdout)

Processing plate='plate16'

Plate has 96 samples (wells)

Data manually specified to drop:
manual_drops: {}

QC thresholds applied to data:
qc_thresholds:
  avg_barcode_counts_per_well: 500
  min_neut_standard_frac_per_well: 0.005
  no_serum_per_viral_barcode_filters:
    min_frac: 0.0001
    max_fold_change: 4
    max_wells: 2
  per_neut_standard_barcode_filters:
    min_frac: 0.005
    max_fold_change: 4
    max_wells: 2
  min_neut_standard_count_per_well: 1000
  min_no_serum_count_per_viral_barcode_well: 100
  max_frac_infectivity_per_viral_barcode_well: 3
  min_dilutions_per_barcode_serum_replicate: 6

Curve-fitting parameters:
curvefit_params:
  frac_infectivity_ceiling: 1
  fixtop:
  - 0.6
  - 1
  fixbottom: 0
  fixslope:
  - 0.8
  - 10

Curve-fitting QC:
curvefit_qc:
  max_frac_infectivity_at_least: 0.0
  goodness_of_fit:
    min_R2: 0.5
    max_RMSD: 0.15
  serum_replicates_ignore_curvefit_qc: []
  barcode_serum_replicates_ignore_curvefit_qc: []

Load the notebook functions:

print(f"Loading {notebook_funcs=}")
%run {notebook_funcs}

Loading notebook_funcs='/home/jbloom/.cache/snakemake/snakemake/source-cache/runtime-cache/tmpfro_n8p6/file/fh/fast/bloom_j/computational_notebooks/jbloom/2025/flu-seqneut-2025/seqneut-pipeline/notebook_funcs.py'

Set up dictionary to keep track of wells, barcodes, well-barcodes, and serum-replicates that are dropped:

qc_drops = {
    "wells": {},
    "barcodes": {},
    "barcode_wells": {},
    "barcode_serum_replicates": {},
    "serum_replicates": {},
}

assert set(manual_drops).issubset(
    qc_drops
), f"{manual_drops.keys()=}, {qc_drops.keys()}"

Statistics on barcode-parsing for each sample

Make interactive chart of the "fates" of the sequencing reads parsed for each sample on the plate.

If most sequencing reads are not "valid barcodes", this could potentially indicate some problem in the sequencing or barcode set you are parsing.

Potential fates are:

• valid barcode: barcode that matches a known virus or neutralization standard, we hope most reads are this.
• invalid barcode: a barcode with proper flanking sequences, but does not match a known virus or neutralization standard. If you have a lot of reads of this type, it is probably a good idea to look at the invalid barcode CSVs (in the ./results/barcode_invalid/ subdirectory created by the pipeline) to see what these invalid barcodes are.
• unparseable barcode: could not parse a barcode from this read as there was not a sequence of the correct length with the appropriate flanking sequence.
• invalid outer flank: if using an outer upstream or downstream region (upstream2 or downstream2 for the illuminabarcodeparser), reads that are otherwise valid except for this outer flank. Typically you would be using upstream2 if you have a plate index embedded in your primer, and reads with this classification correspond to a different index than the one for this plate.
• low quality barcode: low-quality or N nucleotides in barcode, could indicate problem with sequencing.
• failed chastity filter: reads that failed the Illumina chastity filter, if these are reported in the FASTQ (they may not be).

Also, if the number of reads per sample is very uneven, that could indicate that you did not do a good job of balancing the different samples in the Illumina sequencing.

fates = (
    pd.concat([pd.read_csv(f).assign(sample=s) for f, s in zip(fate_csvs, samples)])
    .merge(samples_df, validate="many_to_one", on="sample")
    .assign(
        fate_counts=lambda x: x.groupby("fate")["count"].transform("sum"),
        sample_well=lambda x: x["sample_noplate"] + " (" + x["well"] + ")",
    )
    .query("fate_counts > 0")[  # only keep fates with at least one count
        ["fate", "count", "well", "serum_replicate", "sample_well", "dilution_factor"]
    ]
)

assert len(fates) == len(fates.drop_duplicates())

serum_replicates = sorted(fates["serum_replicate"].unique())
sample_wells = list(
    fates.sort_values(["serum_replicate", "dilution_factor"])["sample_well"]
)


serum_selection = alt.selection_point(
    fields=["serum_replicate"],
    bind=alt.binding_select(
        options=[None] + serum_replicates,
        labels=["all"] + serum_replicates,
        name="serum",
    ),
)

fates_chart = (
    alt.Chart(fates)
    .add_params(serum_selection)
    .transform_filter(serum_selection)
    .encode(
        alt.X("count", scale=alt.Scale(nice=False, padding=3)),
        alt.Y(
            "sample_well",
            title=None,
            sort=sample_wells,
        ),
        alt.Color("fate", sort=sorted(fates["fate"].unique(), reverse=True)),
        alt.Order("fate", sort="descending"),
        tooltip=fates.columns.tolist(),
    )
    .mark_bar(height={"band": 0.85})
    .properties(
        height=alt.Step(10),
        width=200,
        title=f"Barcode parsing for {plate}",
    )
    .configure_axis(grid=False)
)

fates_chart

Read barcode counts and apply manually specified drops

Read the counts per barcode:

# get barcode counts
counts = (
    pd.concat([pd.read_csv(c).assign(sample=s) for c, s in zip(count_csvs, samples)])
    .merge(samples_df, validate="many_to_one", on="sample")
    .drop(columns=["replicate", "plate", "fastq"])
    .assign(sample_well=lambda x: x["sample_noplate"] + " (" + x["well"] + ")")
)

# classify barcodes as viral or neut standard
barcode_class = pd.concat(
    [
        pd.DataFrame(viral_barcodes).assign(neut_standard=False),
        pd.DataFrame(neut_standard_barcodes).assign(neut_standard=True, strain=pd.NA),
    ],
    ignore_index=True,
)

# merge counts and classification of barcodes
assert set(counts["barcode"]) == set(barcode_class["barcode"])
counts = counts.merge(barcode_class, on="barcode", validate="many_to_one")
assert set(sample_wells) == set(counts["sample_well"])
assert set(serum_replicates) == set(counts["serum_replicate"])

Apply any manually specified data drops:

for filter_type, filter_drops in manual_drops.items():
    print(f"\nDropping {len(filter_drops)} {filter_type} specified in manual_drops")
    assert filter_type in qc_drops
    qc_drops[filter_type].update(
        {w: "manual_drop" for w in filter_drops if not isinstance(w, list)}
    )
    if filter_type == "barcode_wells":
        counts = counts[
            ~counts.assign(
                barcode_well=lambda x: x.apply(
                    lambda r: (r["barcode"], r["well"]), axis=1
                )
            )["barcode_well"].isin(qc_drops[filter_type])
        ]
    elif filter_type == "barcode_serum_replicates":
        counts = counts[
            ~counts.assign(
                barcode_serum_replicate=lambda x: x.apply(
                    lambda r: (r["barcode"], r["serum_replicate"]), axis=1
                )
            )["barcode_serum_replicate"].isin(qc_drops[filter_type])
        ]
    elif filter_type == "wells":
        counts = counts[~counts["well"].isin(qc_drops[filter_type])]
    elif filter_type == "barcodes":
        counts = counts[~counts["barcode"].isin(qc_drops[filter_type])]
    elif filter_type == "serum_replicates":
        counts = counts[~counts["serum_replicate"].isin(qc_drops[filter_type])]
    elif filter_type == "barcode_serum_replicates":
        counts = counts[~counts["barcode_serum_replicate"].isin(qc_drops[filter_type])]
    else:
        assert filter_type in set(counts.columns)
        counts = counts[~counts[filter_type].isin(qc_drops[filter_type])]

Average counts per barcode in each well

Plot average counts per barcode. If a sample has inadequate barcode counts, it may not have good enough statistics for accurate analysis, and a QC-threshold is applied:

avg_barcode_counts = (
    counts.groupby(
        ["well", "serum_replicate", "sample_well"],
        dropna=False,
        as_index=False,
    )
    .aggregate(avg_count=pd.NamedAgg("count", "mean"))
    .assign(
        fails_qc=lambda x: (
            x["avg_count"] < qc_thresholds["avg_barcode_counts_per_well"]
        ),
    )
)

avg_barcode_counts_chart = (
    alt.Chart(avg_barcode_counts)
    .add_params(serum_selection)
    .transform_filter(serum_selection)
    .encode(
        alt.X(
            "avg_count",
            title="average barcode counts per well",
            scale=alt.Scale(nice=False, padding=3),
        ),
        alt.Y("sample_well", sort=sample_wells),
        alt.Color(
            "fails_qc",
            title=f"fails {qc_thresholds['avg_barcode_counts_per_well']=}",
            legend=alt.Legend(titleLimit=500),
        ),
        tooltip=[
            alt.Tooltip(c, format=".3g") if avg_barcode_counts[c].dtype == float else c
            for c in avg_barcode_counts.columns
        ],
    )
    .mark_bar(height={"band": 0.85})
    .properties(
        height=alt.Step(10),
        width=250,
        title=f"Average barcode counts per well for {plate}",
    )
    .configure_axis(grid=False)
)

display(avg_barcode_counts_chart)

# drop wells failing QC
avg_barcode_counts_per_well_drops = list(avg_barcode_counts.query("fails_qc")["well"])
print(
    f"\nDropping {len(avg_barcode_counts_per_well_drops)} wells for failing "
    f"{qc_thresholds['avg_barcode_counts_per_well']=}: "
    + str(avg_barcode_counts_per_well_drops)
)
qc_drops["wells"].update(
    {w: "avg_barcode_counts_per_well" for w in avg_barcode_counts_per_well_drops}
)
counts = counts[~counts["well"].isin(qc_drops["wells"])]

Dropping 0 wells for failing qc_thresholds['avg_barcode_counts_per_well']=500: []

Fraction of counts from neutralization standard

Determine the fraction of counts from the neutralization standard in each sample, and make sure this fraction passess the QC threshold.

neut_standard_fracs = (
    counts.assign(
        neut_standard_count=lambda x: x["count"] * x["neut_standard"].astype(int)
    )
    .groupby(
        ["well", "serum_replicate", "sample_well"],
        dropna=False,
        as_index=False,
    )
    .aggregate(
        total_count=pd.NamedAgg("count", "sum"),
        neut_standard_count=pd.NamedAgg("neut_standard_count", "sum"),
    )
    .assign(
        neut_standard_frac=lambda x: x["neut_standard_count"] / x["total_count"],
        fails_qc=lambda x: (
            x["neut_standard_frac"] < qc_thresholds["min_neut_standard_frac_per_well"]
        ),
    )
)

neut_standard_fracs_chart = (
    alt.Chart(neut_standard_fracs)
    .add_params(serum_selection)
    .transform_filter(serum_selection)
    .encode(
        alt.X(
            "neut_standard_frac",
            title="frac counts from neutralization standard per well",
            scale=alt.Scale(nice=False, padding=3),
        ),
        alt.Y("sample_well", sort=sample_wells),
        alt.Color(
            "fails_qc",
            title=f"fails {qc_thresholds['min_neut_standard_frac_per_well']=}",
            legend=alt.Legend(titleLimit=500),
        ),
        tooltip=[
            alt.Tooltip(c, format=".3g") if neut_standard_fracs[c].dtype == float else c
            for c in neut_standard_fracs.columns
        ],
    )
    .mark_bar(height={"band": 0.85})
    .properties(
        height=alt.Step(10),
        width=250,
        title=f"Neutralization-standard fracs per well for {plate}",
    )
    .configure_axis(grid=False)
    .configure_legend(titleLimit=1000)
)

display(neut_standard_fracs_chart)

# drop wells failing QC
min_neut_standard_frac_per_well_drops = list(
    neut_standard_fracs.query("fails_qc")["well"]
)
print(
    f"\nDropping {len(min_neut_standard_frac_per_well_drops)} wells for failing "
    f"{qc_thresholds['min_neut_standard_frac_per_well']=}: "
    + str(min_neut_standard_frac_per_well_drops)
)
qc_drops["wells"].update(
    {
        w: "min_neut_standard_frac_per_well"
        for w in min_neut_standard_frac_per_well_drops
    }
)
counts = counts[~counts["well"].isin(qc_drops["wells"])]

Dropping 0 wells for failing qc_thresholds['min_neut_standard_frac_per_well']=0.005: []

Consistency and minimum fractions for barcodes

We examine the fraction of counts attributable to each barcode. We do this splitting the data two ways:

1. Looking at all viral (but not neut-standard) barcodes only for the no-serum samples (wells).

2. Looking at just the neut-standard barcodes for all samples (wells).

The reasons is that if the experiment is set up perfectly, these fractions should be the same across all samples for each barcode. (We do not expect viral barcodes to have consistent fractions across no-serum samples as they will be neutralized differently depending on strain).

We plot these fractions in interactive plots (you can mouseover points and zoom) so you can identify barcodes that fail the expected consistency QC thresholds.

We also make sure the barcodes meet specified QC minimum thresholds for all samples, and flag any that do not.

barcode_selection = alt.selection_point(fields=["barcode"], on="mouseover", empty=False)

# look at all samples for neut standard barcodes, or no-serum samples for all barcodes
for is_neut_standard, df in counts.groupby("neut_standard"):
    if is_neut_standard:
        print(
            f"\n\n{'=' * 89}\nAnalyzing neut-standard barcodes from all samples (wells)"
        )
        qc_name = "per_neut_standard_barcode_filters"
    else:
        print(f"\n\n{'=' * 89}\nAnalyzing all barcodes from no-serum samples (wells)")
        qc_name = "no_serum_per_viral_barcode_filters"
        df = df.query("serum == 'none'")

    df = df.assign(
        sample_counts=lambda x: x.groupby("sample")["count"].transform("sum"),
        count_frac=lambda x: x["count"] / x["sample_counts"],
        median_count_frac=lambda x: x.groupby("barcode")["count_frac"].transform(
            "median"
        ),
        fold_change_from_median=lambda x: numpy.where(
            x["count_frac"] > x["median_count_frac"],
            x["count_frac"] / x["median_count_frac"],
            x["median_count_frac"] / x["count_frac"],
        ),
    )[
        [
            "barcode",
            "count",
            "sample_well",
            "count_frac",
            "fold_change_from_median",
        ]
        + ([] if is_neut_standard else ["strain"])
    ]

    # barcode fails QC if fails in sufficient wells
    qc = qc_thresholds[qc_name]
    print(f"Apply QC {qc_name}: {qc}\n")
    fails_qc = (
        df.assign(
            fails_qc=lambda x: ~(
                (x["count_frac"] >= qc["min_frac"])
                & (x["fold_change_from_median"] <= qc["max_fold_change"])
            ),
        )
        .groupby("barcode", as_index=False)
        .aggregate(n_wells_fail_qc=pd.NamedAgg("fails_qc", "sum"))
        .assign(fails_qc=lambda x: x["n_wells_fail_qc"] >= qc["max_wells"])[
            ["barcode", "fails_qc"]
        ]
    )
    df = df.merge(fails_qc, on="barcode", validate="many_to_one")

    # make chart
    evenness_chart = (
        alt.Chart(df)
        .add_params(barcode_selection)
        .encode(
            alt.X(
                "count_frac",
                title=(
                    "barcode's fraction of neut standard counts"
                    if is_neut_standard
                    else "barcode's fraction of non-neut standard counts"
                ),
                scale=alt.Scale(nice=False, padding=5),
            ),
            alt.Y("sample_well", sort=sample_wells),
            alt.Fill(
                "fails_qc",
                title=f"fails {qc_name}",
                legend=alt.Legend(titleLimit=500),
            ),
            strokeWidth=alt.condition(barcode_selection, alt.value(2), alt.value(0)),
            size=alt.condition(barcode_selection, alt.value(60), alt.value(35)),
            tooltip=[
                alt.Tooltip(c, format=".2g") if df[c].dtype == float else c
                for c in df.columns
            ],
        )
        .mark_circle(fillOpacity=0.45, stroke="black", strokeOpacity=1)
        .properties(
            height=alt.Step(10),
            width=300,
            title=alt.TitleParams(
                (
                    f"{plate} all samples, neut-standard barcodes"
                    if is_neut_standard
                    else f"{plate} no-serum samples, all barcodes"
                ),
                subtitle="x-axis is zoomable (use mouse scroll/pan)",
            ),
        )
        .configure_axis(grid=False)
        .configure_legend(titleLimit=1000)
        .interactive()
    )

    display(evenness_chart)

    # drop barcodes failing QC
    barcode_drops = list(fails_qc.query("fails_qc")["barcode"])
    print(
        f"\nDropping {len(barcode_drops)} barcodes for failing {qc=}: {barcode_drops}"
    )
    qc_drops["barcodes"].update(
        {bc: "min_neut_standard_frac_per_well" for bc in barcode_drops}
    )
    counts = counts[~counts["barcode"].isin(qc_drops["barcodes"])]

=========================================================================================
Analyzing all barcodes from no-serum samples (wells)
Apply QC no_serum_per_viral_barcode_filters: {'min_frac': 0.0001, 'max_fold_change': 4, 'max_wells': 2}

Dropping 3 barcodes for failing qc={'min_frac': 0.0001, 'max_fold_change': 4, 'max_wells': 2}: ['AGTCCTATCCTCAAAT', 'CTCTTACGCTCCTACG', 'GATTCAGATGCCCACC']


=========================================================================================
Analyzing neut-standard barcodes from all samples (wells)
Apply QC per_neut_standard_barcode_filters: {'min_frac': 0.005, 'max_fold_change': 4, 'max_wells': 2}

Dropping 0 barcodes for failing qc={'min_frac': 0.005, 'max_fold_change': 4, 'max_wells': 2}: []

Compute fraction infectivity

The fraction infectivity for viral barcode $v_b$ in sample $s$ is computed as: $$ F_{v_b,s} = \frac{c_{v_b,s} / \left(\sum_{n_b} c_{n_b,s}\right)}{{\rm median}_{s_0}\left[ c_{v_b,s_0} / \left(\sum_{n_b} c_{n_b,s_0}\right)\right]} $$ where

• $c_{v_b,s}$ is the counts of viral barcode $v_b$ in sample $s$.
• $\sum_{n_b} c_{n_b,s}$ is the sum of the counts for all neutralization standard barcodes $n_b$ for sample $s$.
• $c_{v_b,s_0}$ is the counts of viral barcode $v_b$ in no-serum sample $s_0$.
• $\sum_{n_b} c_{n_b,s_0}$ is the sum of the counts for all neutralization standard barcodes $n_b$ for no-serum sample $s_0$.
• ${\rm median}_{s_0}\left[ c_{v_b,s_0} / \left(\sum_{n_b} c_{n_b,s_0}\right)\right]$ is the median taken across all no-serum samples of the counts of viral barcode $v_b$ versus the total counts for all neutralization standard barcodes.

First, compute the total neutralization-standard counts for each sample (well). Plot these, and drop any wells that do not meet the QC threshold.

neut_standard_counts = (
    counts.query("neut_standard")
    .groupby(
        ["well", "serum_replicate", "sample_well", "dilution_factor"],
        dropna=False,
        as_index=False,
    )
    .aggregate(neut_standard_count=pd.NamedAgg("count", "sum"))
    .assign(
        fails_qc=lambda x: (
            x["neut_standard_count"] < qc_thresholds["min_neut_standard_count_per_well"]
        ),
    )
)

neut_standard_counts_chart = (
    alt.Chart(neut_standard_counts)
    .add_params(serum_selection)
    .transform_filter(serum_selection)
    .encode(
        alt.X(
            "neut_standard_count",
            title="counts from neutralization standard",
            scale=alt.Scale(nice=False, padding=3),
        ),
        alt.Y("sample_well", sort=sample_wells),
        alt.Color(
            "fails_qc",
            title=f"fails {qc_thresholds['min_neut_standard_count_per_well']=}",
            legend=alt.Legend(titleLimit=500),
        ),
        tooltip=[
            (
                alt.Tooltip(c, format=".3g")
                if neut_standard_counts[c].dtype == float
                else c
            )
            for c in neut_standard_counts.columns
        ],
    )
    .mark_bar(height={"band": 0.85})
    .properties(
        height=alt.Step(10),
        width=250,
        title=f"Neutralization-standard counts for {plate}",
    )
    .configure_axis(grid=False)
    .configure_legend(titleLimit=1000)
)

display(neut_standard_counts_chart)

# drop wells failing QC
min_neut_standard_count_per_well_drops = list(
    neut_standard_counts.query("fails_qc")["well"]
)
print(
    f"\nDropping {len(min_neut_standard_count_per_well_drops)} wells for failing "
    f"{qc_thresholds['min_neut_standard_count_per_well']=}: "
    + str(min_neut_standard_count_per_well_drops)
)
qc_drops["wells"].update(
    {
        w: "min_neut_standard_count_per_well"
        for w in min_neut_standard_count_per_well_drops
    }
)
neut_standard_counts = neut_standard_counts[
    ~neut_standard_counts["well"].isin(qc_drops["wells"])
]
counts = counts[~counts["well"].isin(qc_drops["wells"])]

Dropping 0 wells for failing qc_thresholds['min_neut_standard_count_per_well']=1000: []

Compute and plot the no-serum sample viral barcode counts and check if they pass the QC filters.

no_serum_counts = (
    counts.query("serum == 'none'")
    .query("not neut_standard")
    .merge(neut_standard_counts, validate="many_to_one")[
        ["barcode", "strain", "well", "sample_well", "count", "neut_standard_count"]
    ]
    .assign(
        fails_qc=lambda x: (
            x["count"] <= qc_thresholds["min_no_serum_count_per_viral_barcode_well"]
        ),
    )
)

strains = sorted(no_serum_counts["strain"].unique())
strain_selection_dropdown = alt.selection_point(
    fields=["strain"],
    bind=alt.binding_select(
        options=[None] + strains,
        labels=["all"] + strains,
        name="virus strain",
    ),
)

# make chart
no_serum_counts_plot_df = no_serum_counts.drop(columns=["well", "neut_standard_count"])
no_serum_counts_chart = (
    alt.Chart(no_serum_counts_plot_df)
    .add_params(barcode_selection, strain_selection_dropdown)
    .transform_filter(strain_selection_dropdown)
    .encode(
        alt.X(
            "count", title="viral barcode count", scale=alt.Scale(nice=False, padding=5)
        ),
        alt.Y("sample_well", sort=sample_wells),
        alt.Fill(
            "fails_qc",
            title=f"fails {qc_thresholds['min_no_serum_count_per_viral_barcode_well']=}",
            legend=alt.Legend(titleLimit=500),
        ),
        strokeWidth=alt.condition(barcode_selection, alt.value(2), alt.value(0)),
        size=alt.condition(barcode_selection, alt.value(60), alt.value(35)),
        tooltip=no_serum_counts_plot_df.columns.tolist(),
    )
    .mark_circle(fillOpacity=0.6, stroke="black", strokeOpacity=1)
    .properties(
        height=alt.Step(10),
        width=400,
        title=f"{plate} viral barcode counts in no-serum samples",
    )
    .configure_axis(grid=False)
    .configure_legend(titleLimit=1000)
    .interactive()
)

display(no_serum_counts_chart)

# drop barcode / wells failing QC
min_no_serum_count_per_viral_barcode_well_drops = list(
    no_serum_counts.query("fails_qc")[["barcode", "well"]].itertuples(
        index=False, name=None
    )
)
print(
    f"\nDropping {len(min_no_serum_count_per_viral_barcode_well_drops)} barcode-wells for failing "
    f"{qc_thresholds['min_no_serum_count_per_viral_barcode_well']=}: "
    + str(min_no_serum_count_per_viral_barcode_well_drops)
)
qc_drops["barcode_wells"].update(
    {
        w: "min_no_serum_count_per_viral_barcode_well"
        for w in min_no_serum_count_per_viral_barcode_well_drops
    }
)
no_serum_counts = no_serum_counts[
    ~no_serum_counts.assign(
        barcode_well=lambda x: x.apply(lambda r: (r["barcode"], r["well"]), axis=1)
    )["barcode_well"].isin(qc_drops["barcode_wells"])
]
counts = counts[
    ~counts.assign(
        barcode_well=lambda x: x.apply(lambda r: (r["barcode"], r["well"]), axis=1)
    )["barcode_well"].isin(qc_drops["barcode_wells"])
]

Dropping 3 barcode-wells for failing qc_thresholds['min_no_serum_count_per_viral_barcode_well']=100: [('TACAAGAGAGGGGTCC', 'A12'), ('CCTTTCTCAAAACATA', 'G12'), ('TACAAGAGAGGGGTCC', 'H12')]

Compute and plot the median ratio of viral barcode count to neut standard counts across no-serum samples. If library composition is equal, all of these values should be similar:

median_no_serum_ratio = (
    no_serum_counts.assign(ratio=lambda x: x["count"] / x["neut_standard_count"])
    .groupby(["barcode", "strain"], as_index=False)
    .aggregate(median_no_serum_ratio=pd.NamedAgg("ratio", "median"))
)

strain_selection = alt.selection_point(fields=["strain"], on="mouseover", empty=False)

median_no_serum_ratio_chart = (
    alt.Chart(median_no_serum_ratio)
    .add_params(strain_selection)
    .encode(
        alt.X(
            "median_no_serum_ratio",
            title="median ratio of counts",
            scale=alt.Scale(nice=False, padding=5),
        ),
        alt.Y(
            "barcode",
            sort=alt.SortField("median_no_serum_ratio", order="descending"),
            axis=alt.Axis(labelFontSize=5),
        ),
        color=alt.condition(strain_selection, alt.value("orange"), alt.value("gray")),
        tooltip=[
            (
                alt.Tooltip(c, format=".3g")
                if median_no_serum_ratio[c].dtype == float
                else c
            )
            for c in median_no_serum_ratio.columns
        ],
    )
    .mark_bar(height={"band": 0.85})
    .properties(
        height=alt.Step(5),
        width=250,
        title=f"{plate} no-serum median ratio viral barcode to neut-standard barcode",
    )
    .configure_axis(grid=False)
    .configure_legend(titleLimit=1000)
)

display(median_no_serum_ratio_chart)

Compute the actual fraction infectivities. We compute both the raw fraction infectivities and the ones with the ceiling applied:

frac_infectivity = (
    counts.query("not neut_standard")
    .query("serum != 'none'")
    .merge(median_no_serum_ratio, validate="many_to_one")
    .merge(neut_standard_counts, validate="many_to_one")
    .assign(
        frac_infectivity_raw=lambda x: (
            (x["count"] / x["neut_standard_count"]) / x["median_no_serum_ratio"]
        ),
        frac_infectivity_ceiling=lambda x: x["frac_infectivity_raw"].clip(
            upper=curvefit_params["frac_infectivity_ceiling"]
        ),
        concentration=lambda x: 1 / x["dilution_factor"],
        plate_barcode=lambda x: x["plate_replicate"] + "-" + x["barcode"],
    )[
        [
            "barcode",
            "plate_barcode",
            "well",
            "strain",
            "serum",
            "serum_replicate",
            "dilution_factor",
            "concentration",
            "frac_infectivity_raw",
            "frac_infectivity_ceiling",
        ]
    ]
)

assert len(
    frac_infectivity.groupby(["serum", "plate_barcode", "dilution_factor"])
) == len(frac_infectivity)
assert frac_infectivity["dilution_factor"].notnull().all()
assert frac_infectivity["frac_infectivity_raw"].notnull().all()
assert frac_infectivity["frac_infectivity_ceiling"].notnull().all()

Plot the fraction infectivities, both the raw values and with the ceiling applied:

frac_infectivity_cols = {
    "frac_infectivity_raw": "raw fraction infectivity",
    "frac_infectivity_ceiling": f"fraction infectivity with ceiling at {curvefit_params['frac_infectivity_ceiling']}",
}

frac_infectivity_chart_df = frac_infectivity.assign(
    fails_qc=lambda x: (
        x["frac_infectivity_raw"]
        > qc_thresholds["max_frac_infectivity_per_viral_barcode_well"]
    ),
)[
    [
        "barcode",
        "strain",
        "well",
        "serum_replicate",
        "dilution_factor",
        "fails_qc",
        *list(frac_infectivity_cols),
    ]
].rename(
    columns=frac_infectivity_cols
)

# some manipulations to shrink data frame plotted by altair below by putting
# them in smaller data frames that are used via transform_lookup
barcode_lookup_df = frac_infectivity[["barcode", "strain"]].drop_duplicates()
assert len(barcode_lookup_df) == barcode_lookup_df["barcode"].nunique()
well_lookup_df = frac_infectivity[
    ["well", "serum_replicate", "dilution_factor"]
].drop_duplicates()
assert len(well_lookup_df) == well_lookup_df["well"].nunique()

frac_infectivity_chart_df = frac_infectivity_chart_df.drop(
    columns=["strain", "serum_replicate", "dilution_factor"]
)

frac_infectivity_chart = (
    alt.Chart(frac_infectivity_chart_df)
    .transform_lookup(
        lookup="barcode",
        from_=alt.LookupData(barcode_lookup_df, key="barcode", fields=["strain"]),
    )
    .transform_lookup(
        lookup="well",
        from_=alt.LookupData(
            well_lookup_df, key="well", fields=["serum_replicate", "dilution_factor"]
        ),
    )
    .transform_fold(
        frac_infectivity_cols.values(), ["ceiling_applied", "frac_infectivity"]
    )
    .add_params(strain_selection_dropdown, barcode_selection)
    .transform_filter(strain_selection_dropdown)
    .encode(
        alt.X(
            "dilution_factor:Q",
            title="dilution factor",
            scale=alt.Scale(nice=False, padding=5, type="log"),
        ),
        alt.Y(
            "frac_infectivity:Q",
            title="fraction infectivity",
            scale=alt.Scale(nice=False, padding=5),
        ),
        alt.Column(
            "ceiling_applied:N",
            sort="descending",
            title=None,
            header=alt.Header(labelFontSize=13, labelFontStyle="bold", labelPadding=2),
        ),
        alt.Row(
            "serum_replicate:N",
            title=None,
            spacing=3,
            header=alt.Header(labelFontSize=13, labelFontStyle="bold"),
        ),
        alt.Detail("barcode"),
        alt.Shape(
            "fails_qc",
            title=f"fails {qc_thresholds['max_frac_infectivity_per_viral_barcode_well']=}",
            legend=alt.Legend(titleLimit=500, orient="bottom"),
        ),
        color=alt.condition(
            barcode_selection, alt.value("black"), alt.value("MediumBlue")
        ),
        strokeWidth=alt.condition(barcode_selection, alt.value(3), alt.value(1)),
        opacity=alt.condition(barcode_selection, alt.value(1), alt.value(0.25)),
        tooltip=[
            (
                alt.Tooltip(c, format=".3g")
                if frac_infectivity_chart_df[c].dtype == float
                else c
            )
            for c in frac_infectivity_chart_df.columns
        ]
        + [
            alt.Tooltip("strain:N"),
            alt.Tooltip("serum_replicate:N"),
            alt.Tooltip("dilution_factor:Q"),
        ],
    )
    .mark_line(point=True)
    .properties(
        height=150,
        width=250,
        title=f"Fraction infectivities for {plate}",
    )
    .interactive(bind_x=False)
    .configure_axis(grid=False)
    .configure_legend(titleLimit=1000)
    .configure_point(size=50)
    .resolve_scale(x="independent", y="independent")
)

display(frac_infectivity_chart)

# drop barcode / wells failing QC
max_frac_infectivity_per_viral_barcode_well_drops = list(
    frac_infectivity_chart_df.query("fails_qc")[["barcode", "well"]]
    .drop_duplicates()
    .itertuples(index=False, name=None)
)
print(
    f"\nDropping {len(max_frac_infectivity_per_viral_barcode_well_drops)} barcode-wells for failing "
    f"{qc_thresholds['max_frac_infectivity_per_viral_barcode_well']=}: "
    + str(max_frac_infectivity_per_viral_barcode_well_drops)
)
qc_drops["barcode_wells"].update(
    {
        w: "max_frac_infectivity_per_viral_barcode_well"
        for w in max_frac_infectivity_per_viral_barcode_well_drops
    }
)
frac_infectivity = frac_infectivity[
    ~frac_infectivity.assign(
        barcode_well=lambda x: x.apply(lambda r: (r["barcode"], r["well"]), axis=1)
    )["barcode_well"].isin(qc_drops["barcode_wells"])
]

Dropping 61 barcode-wells for failing qc_thresholds['max_frac_infectivity_per_viral_barcode_well']=3: [('AGATCCACCCTATAGT', 'F1'), ('CACCAATCTTCGAACT', 'F2'), ('GAGGGGATACGTCACC', 'G2'), ('GCATGGAACTAACTCC', 'D3'), ('GATCACGCAGAAAAAG', 'E3'), ('AGGACTATAGTTGGCA', 'F3'), ('CACCAATCTTCGAACT', 'F3'), ('ATGGTTTTACGTCCAT', 'H3'), ('AGATCCACCCTATAGT', 'D4'), ('CGCGACACCCTTCCGG', 'E4'), ('TATTAAGAGAAGTGCG', 'E4'), ('TCGAGTTAATATGCGC', 'E4'), ('GTGGTATCAAGCCGGG', 'G4'), ('CCGGATAAATCAGAAC', 'G4'), ('GAAAGTCCCTATGATG', 'G4'), ('ATGGTTTTACGTCCAT', 'G4'), ('GCAGCGTGCCGGTCAT', 'H4'), ('CACCAATCTTCGAACT', 'B5'), ('CGCGACACCCTTCCGG', 'E5'), ('CGTGACCCCCTCCAAC', 'E5'), ('TCGCTTCAACTAAAAA', 'E5'), ('ATGGTTTTACGTCCAT', 'E5'), ('CACCTAGGATCGCACT', 'G5'), ('AGATCCACCCTATAGT', 'F6'), ('CACCAATCTTCGAACT', 'G6'), ('GCCGCTGCGGCGTGTG', 'H6'), ('CCTTTCTCAAAACATA', 'E7'), ('GACCCCTTGTAAGATG', 'F7'), ('AGGTTCAGACTCTTGC', 'G7'), ('GACCCCTTGTAAGATG', 'G7'), ('TCTCAGCTCTTAGCCG', 'H7'), ('GACCCCTTGTAAGATG', 'E8'), ('AACCGTACCGCGTTTA', 'G8'), ('CGAAACACGTCCCAGT', 'G8'), ('CCTTTCTCAAAACATA', 'G8'), ('GTGCATCCTAGTGACG', 'G8'), ('AGATCCACCCTATAGT', 'H8'), ('ATAACTGAGGGCATTG', 'E9'), ('AATGCGAGCATGTCAA', 'E9'), ('GTGCATCCTAGTGACG', 'E9'), ('TCGAGTTAATATGCGC', 'E9'), ('AATGCGAGCATGTCAA', 'G9'), ('CACTAGATGTACAGTC', 'G9'), ('GAAATCCCCAAATAAC', 'G9'), ('AGATCCACCCTATAGT', 'G9'), ('TCGAGTTAATATGCGC', 'G9'), ('CACCAATCTTCGAACT', 'G9'), ('CCTTTCTCAAAACATA', 'G9'), ('AAAGCTCTTTTCGTTC', 'H9'), ('GCATGGAACTAACTCC', 'H9'), ('TAATAAGCCAGCAAGA', 'H9'), ('CTTACAGAATACTAGA', 'H9'), ('CGACTCCACGGACGCC', 'H9'), ('GACCCCTTGTAAGATG', 'H9'), ('ATGGTTTTACGTCCAT', 'D10'), ('AATGCGAGCATGTCAA', 'E10'), ('GCCGGCGTTAGTGTCA', 'F10'), ('CCTTTCTCAAAACATA', 'F10'), ('ACAGTACGATCTACGC', 'G10'), ('TTTATATCGAGATTCA', 'E11'), ('TCGATTACTAGCCGGA', 'G11')]

Check how many dilutions we have per barcode / serum-replicate:

n_dilutions = (
    frac_infectivity.groupby(["serum_replicate", "strain", "barcode"], as_index=False)
    .aggregate(**{"number of dilutions": pd.NamedAgg("dilution_factor", "nunique")})
    .assign(
        fails_qc=lambda x: (
            x["number of dilutions"]
            < qc_thresholds["min_dilutions_per_barcode_serum_replicate"]
        ),
    )
)

n_dilutions_chart = (
    alt.Chart(n_dilutions)
    .add_params(barcode_selection)
    .encode(
        alt.X("number of dilutions", scale=alt.Scale(nice=False, padding=4)),
        alt.Y("strain", title=None),
        alt.Column(
            "serum_replicate",
            title=None,
            header=alt.Header(labelFontSize=12, labelFontStyle="bold", labelPadding=0),
        ),
        alt.Fill(
            "fails_qc",
            title=f"fails {qc_thresholds['min_dilutions_per_barcode_serum_replicate']=}",
            legend=alt.Legend(titleLimit=500, orient="bottom"),
        ),
        strokeWidth=alt.condition(barcode_selection, alt.value(2), alt.value(0)),
        size=alt.condition(barcode_selection, alt.value(55), alt.value(35)),
        tooltip=[
            alt.Tooltip(c, format=".3g") if n_dilutions[c].dtype == float else c
            for c in n_dilutions.columns
        ],
    )
    .mark_circle(stroke="black", strokeOpacity=1, fillOpacity=0.45)
    .properties(
        height=alt.Step(10),
        width=120,
        title=alt.TitleParams(
            "number of dilutions for each barcode for each serum-replicate", dy=-2
        ),
    )
)

display(n_dilutions_chart)

# drop barcode / serum-replicates failing QC
min_dilutions_per_barcode_serum_replicate_drops = list(
    n_dilutions.query("fails_qc")[["barcode", "serum_replicate"]].itertuples(
        index=False, name=None
    )
)
print(
    f"\nDropping {len(min_dilutions_per_barcode_serum_replicate_drops)} barcode/serum-replicates for failing "
    f"{qc_thresholds['min_dilutions_per_barcode_serum_replicate']=}: "
    + str(min_dilutions_per_barcode_serum_replicate_drops)
)
qc_drops["barcode_serum_replicates"].update(
    {
        w: "min_dilutions_per_barcode_serum_replicate"
        for w in min_dilutions_per_barcode_serum_replicate_drops
    }
)
frac_infectivity = frac_infectivity[
    ~frac_infectivity.assign(
        barcode_serum_replicate=lambda x: x.apply(
            lambda r: (r["barcode"], r["serum_replicate"]), axis=1
        )
    )["barcode_serum_replicate"].isin(qc_drops["barcode_serum_replicates"])
]

Dropping 0 barcode/serum-replicates for failing qc_thresholds['min_dilutions_per_barcode_serum_replicate']=6: []

Fit neutralization curves without applying QC to curves

First fit curves to all serum replicates, then we will apply QC on the curve fits. Note that the fitting is done to the fraction infectivities with the ceiling:

fits_noqc = neutcurve.CurveFits(
    frac_infectivity.rename(
        columns={
            "frac_infectivity_ceiling": "fraction infectivity",
            "concentration": "serum concentration",
        }
    ),
    conc_col="serum concentration",
    fracinf_col="fraction infectivity",
    virus_col="strain",
    serum_col="serum_replicate",
    replicate_col="barcode",
    fixtop=curvefit_params["fixtop"],
    fixbottom=curvefit_params["fixbottom"],
    fixslope=curvefit_params["fixslope"],
)

Determine which fits fail the curve fitting QC, and plot them. Note the plot indicates as failing QC any barcode / serum-replicate that fails, even if we are also specified to ignore the QC for that one (so it will not be removed later):

goodness_of_fit = curvefit_qc["goodness_of_fit"]

fit_params_noqc = (
    frac_infectivity.groupby(["serum_replicate", "barcode"], as_index=False)
    .aggregate(max_frac_infectivity=pd.NamedAgg("frac_infectivity_ceiling", "max"))
    .merge(
        fits_noqc.fitParams(average_only=False, no_average=True)[
            ["serum", "virus", "replicate", "r2", "rmsd"]
        ].rename(columns={"serum": "serum_replicate", "replicate": "barcode"}),
        validate="one_to_one",
    )
    .assign(
        fails_max_frac_infectivity_at_least=lambda x: (
            x["max_frac_infectivity"] < curvefit_qc["max_frac_infectivity_at_least"]
        ),
        fails_goodness_of_fit=lambda x: (
            (x["r2"] < goodness_of_fit["min_R2"])
            & (x["rmsd"] > goodness_of_fit["max_RMSD"])
        ),
        fails_qc=lambda x: (
            x["fails_max_frac_infectivity_at_least"] | x["fails_goodness_of_fit"]
        ),
        ignore_qc=lambda x: x.apply(
            lambda r: (
                (
                    r["serum_replicate"]
                    in curvefit_qc["serum_replicates_ignore_curvefit_qc"]
                )
                or (
                    (r["barcode"], r["serum_replicate"])
                    in curvefit_qc["barcode_serum_replicates_ignore_curvefit_qc"]
                )
            ),
            axis=1,
        ),
    )
)

print(f"Plotting barcode / serum-replicates that fail {curvefit_qc=}\n")

fit_params_noqc_base_chart = alt.Chart(fit_params_noqc).add_params(barcode_selection)
fit_params_noqc_chart = []
for prop, col in [
    ("max frac infectivity", "max_frac_infectivity"),
    ("curve fit R2", "r2"),
    ("curve fit RMSD", "rmsd"),
]:
    fit_params_noqc_chart.append(
        fit_params_noqc_base_chart.encode(
            alt.X(col, title=prop, scale=alt.Scale(nice=False, padding=4)),
            alt.Y("virus", title=None),
            alt.Fill("fails_qc"),
            alt.Column(
                "serum_replicate",
                title=None,
                header=alt.Header(
                    labelFontSize=12, labelFontStyle="bold", labelPadding=0
                ),
            ),
            strokeWidth=alt.condition(barcode_selection, alt.value(2), alt.value(0)),
            size=alt.condition(barcode_selection, alt.value(55), alt.value(35)),
            tooltip=[
                alt.Tooltip(c, format=".3g") if fit_params_noqc[c].dtype == float else c
                for c in fit_params_noqc.columns
            ],
        )
        .mark_circle(stroke="black", strokeOpacity=1, fillOpacity=0.55)
        .properties(
            height=alt.Step(10),
            width=90,
            title=alt.TitleParams(f"{prop} for each barcode serum-replicate", dy=-2),
        )
    )

alt.vconcat(*fit_params_noqc_chart)

Plotting barcode / serum-replicates that fail curvefit_qc={'max_frac_infectivity_at_least': 0.0, 'goodness_of_fit': {'min_R2': 0.5, 'max_RMSD': 0.15}, 'serum_replicates_ignore_curvefit_qc': [], 'barcode_serum_replicates_ignore_curvefit_qc': []}

Now plot curves for all virus vs serum-replicates that have a barcode that fails any of the QC. In these plots, the suffix on the barcode name in the color key indicates if it passed or failed QC:

barcode_serum_replicates_fail_qc = fit_params_noqc.query("fails_qc").reset_index(
    drop=True
)
print(f"Here are barcode / serum-replicates that fail {curvefit_qc=}")
display(barcode_serum_replicates_fail_qc)

if len(barcode_serum_replicates_fail_qc):
    print(
        "\nCurves for virus vs serum-replicates with at least one failed barcode."
        "\nColor key labels indicate if barcodes failed or passed QC."
    )
    plots = {}
    ncol = 6
    for iplot, (serum, virus, failed_barcodes) in enumerate(
        barcode_serum_replicates_fail_qc.groupby(
            ["serum_replicate", "virus"], as_index=False
        )
        .aggregate(barcodes=pd.NamedAgg("barcode", list))
        .itertuples(index=False)
    ):
        passed_barcodes = [
            bc
            for bc in fits_noqc.replicates[serum, virus]
            if (bc not in failed_barcodes) and (bc != "average")
        ]
        curvelist = []
        assert len(CBMARKERS) >= len(failed_barcodes + passed_barcodes)
        assert len(CBPALETTE) >= len(failed_barcodes + passed_barcodes)
        for replicate, marker, color in zip(
            failed_barcodes + passed_barcodes, CBMARKERS, CBPALETTE
        ):
            curvelist.append(
                {
                    "serum": serum,
                    "virus": virus,
                    "replicate": replicate,
                    "label": replicate
                    + ("-fail" if replicate in failed_barcodes else "-pass"),
                    "color": color,
                    "marker": marker,
                }
            )
        plots[iplot // ncol, iplot % ncol] = (f"{serum} vs {virus}", curvelist)

    fig, _ = fits_noqc.plotGrid(
        plots,
        attempt_shared_legend=False,
        legendfontsize=8,
        titlesize=9,
        ticksize=10,
        draw_in_bounds=True,
    )
    display_curve_fig(fig, curve_display_method)
    plt.close(fig)

Here are barcode / serum-replicates that fail curvefit_qc={'max_frac_infectivity_at_least': 0.0, 'goodness_of_fit': {'min_R2': 0.5, 'max_RMSD': 0.15}, 'serum_replicates_ignore_curvefit_qc': [], 'barcode_serum_replicates_ignore_curvefit_qc': []}

	serum_replicate	barcode	max_frac_infectivity	virus	r2	rmsd	fails_max_frac_infectivity_at_least	fails_goodness_of_fit	fails_qc	ignore_qc
0	NIID_1	ACACGGGTTGGCTGTA	1.000000	A/Iowa/123/2024_H1N1	1.305183e-01	0.172802	False	True	True	False
1	NIID_10	AAAGACCTTTAACTCT	1.000000	A/Singapore/INFIMH-16-0019/2016X-307A_H3N2	2.145769e-01	0.196922	False	True	True	False
2	NIID_10	AACCACCCCAGAGATG	1.000000	A/Kansas/14/2017_H3N2	8.193451e-03	0.195628	False	True	True	False
3	NIID_10	ATTTACTCATTATACG	1.000000	A/HongKong/4801/2014egg_H3N2	4.496891e-01	0.174833	False	True	True	False
4	NIID_10	GTAAGCTTCATGGAGT	0.986421	A/Switzerland/860423897313/2023_H3N2	-2.220446e-16	0.179166	False	True	True	False
5	NIID_11	AGACCATCGCACCCAA	1.000000	A/Thailand/8/2022_H3N2	4.245022e-01	0.248223	False	True	True	False
6	NIID_11	AGTGTTGGCTTGGTTA	0.951794	A/Pennsylvania/288/2024_H3N2	1.705239e-01	0.156795	False	True	True	False
7	NIID_11	AGTTGGGGTCTCCCTT	1.000000	A/Amapa/021563-IEC/2024_H3N2	4.826356e-01	0.168506	False	True	True	False
8	NIID_11	CCCTTTACGGATCTCT	1.000000	A/CoteD'Ivoire/4448/2024_H3N2	3.542541e-01	0.196659	False	True	True	False
9	NIID_11	CCGCATTAGCGGGAGG	0.865538	A/Maldives/2186/2024_H3N2	2.137936e-01	0.181612	False	True	True	False
10	NIID_11	CGGTCGGGACTCATCT	1.000000	A/Utah/94/2024_H3N2	4.770573e-01	0.172741	False	True	True	False
11	NIID_11	CGTACGTATGTCCCAG	1.000000	A/Thailand/8/2022_H3N2	4.364622e-01	0.213534	False	True	True	False
12	NIID_11	CTTTTCTAGTACGCTT	1.000000	A/Nevada/216/2024_H3N2	4.785826e-01	0.181638	False	True	True	False
13	NIID_11	GAAGTGCTGCTGAAGT	1.000000	A/Croatia/10136RV/2023-egg_H3N2	4.912441e-01	0.219136	False	True	True	False
14	NIID_11	GCCGCTGCGGCGTGTG	1.000000	A/Norway/12374/2023_H3N2	3.303767e-01	0.182171	False	True	True	False
15	NIID_11	GTAAGCTTCATGGAGT	1.000000	A/Switzerland/860423897313/2023_H3N2	3.515861e-01	0.221894	False	True	True	False
16	NIID_4	ACCCCCGGAGCTTGGC	1.000000	A/Lisboa/188/2023_H1N1	1.393681e-01	0.273664	False	True	True	False
17	NIID_4	AGATCCACCCTATAGT	1.000000	A/Bangkok/P176/2025_H1N1	1.028872e-01	0.156842	False	True	True	False
18	NIID_5	CTGAACTTGTCGATAT	1.000000	A/Wisconsin/67/2022_H1N1	0.000000e+00	0.176098	False	True	True	False
19	NIID_5	GCCATTTACTGAAGGG	1.000000	A/Mato_Grosso_do_Sul/518/2025_H3N2	4.112566e-01	0.185369	False	True	True	False
20	NIID_6	ATTAGATTATAACGTA	1.000000	A/Cambodia/e0826360/2020_H3N2	3.096873e-01	0.175926	False	True	True	False
21	NIID_6	TAAAAAGCCTCCATGA	1.000000	A/HongKong/45/2019_H3N2	0.000000e+00	0.179638	False	True	True	False
22	NIID_7	CCCCCGCTGTTTAAAA	1.000000	A/Washington/284/2024_H3N2	1.872122e-01	0.195055	False	True	True	False
23	NIID_7	GCCTTTGCGCGCAGTC	1.000000	A/Badajoz/18680568/2025_H3N2	4.935943e-01	0.206760	False	True	True	False
24	NIID_7	TTGACTCACCGAATAA	1.000000	A/Cambodia/e0826360/2020_H3N2	0.000000e+00	0.161294	False	True	True	False
25	NIID_8	GTGCATCCTAGTGACG	1.000000	A/Vermont/05/2025_H1N1	4.212932e-01	0.251170	False	True	True	False
26	NIID_9	CAATTCGCCGTTCCCC	1.000000	A/Darwin/6/2021_H3N2	1.817335e-01	0.216808	False	True	True	False

Curves for virus vs serum-replicates with at least one failed barcode.
Color key labels indicate if barcodes failed or passed QC.

# drop barcode / serum-replicates failing QC
for qc_filter in ["max_frac_infectivity_at_least", "goodness_of_fit"]:
    fits_qc_drops = list(
        fit_params_noqc.query(f"fails_{qc_filter} and (not ignore_qc)")[
            ["barcode", "serum_replicate"]
        ].itertuples(index=False, name=None)
    )
    print(
        f"\nDropping {len(fits_qc_drops)} barcode/serum-replicates for failing "
        f"{qc_filter}={curvefit_qc[qc_filter]}: " + str(fits_qc_drops)
    )
    qc_drops["barcode_serum_replicates"].update({w: qc_filter for w in fits_qc_drops})
    frac_infectivity = frac_infectivity[
        ~frac_infectivity.assign(
            barcode_serum_replicate=lambda x: x.apply(
                lambda r: (r["barcode"], r["serum_replicate"]), axis=1
            )
        )["barcode_serum_replicate"].isin(qc_drops["barcode_serum_replicates"])
    ]
    fit_params_noqc = fit_params_noqc[
        ~fit_params_noqc.assign(
            barcode_serum_replicate=lambda x: x.apply(
                lambda r: (r["barcode"], r["serum_replicate"]), axis=1
            )
        )["barcode_serum_replicate"].isin(qc_drops["barcode_serum_replicates"])
    ]

Dropping 0 barcode/serum-replicates for failing max_frac_infectivity_at_least=0.0: []

Dropping 27 barcode/serum-replicates for failing goodness_of_fit={'min_R2': 0.5, 'max_RMSD': 0.15}: [('ACACGGGTTGGCTGTA', 'NIID_1'), ('AAAGACCTTTAACTCT', 'NIID_10'), ('AACCACCCCAGAGATG', 'NIID_10'), ('ATTTACTCATTATACG', 'NIID_10'), ('GTAAGCTTCATGGAGT', 'NIID_10'), ('AGACCATCGCACCCAA', 'NIID_11'), ('AGTGTTGGCTTGGTTA', 'NIID_11'), ('AGTTGGGGTCTCCCTT', 'NIID_11'), ('CCCTTTACGGATCTCT', 'NIID_11'), ('CCGCATTAGCGGGAGG', 'NIID_11'), ('CGGTCGGGACTCATCT', 'NIID_11'), ('CGTACGTATGTCCCAG', 'NIID_11'), ('CTTTTCTAGTACGCTT', 'NIID_11'), ('GAAGTGCTGCTGAAGT', 'NIID_11'), ('GCCGCTGCGGCGTGTG', 'NIID_11'), ('GTAAGCTTCATGGAGT', 'NIID_11'), ('ACCCCCGGAGCTTGGC', 'NIID_4'), ('AGATCCACCCTATAGT', 'NIID_4'), ('CTGAACTTGTCGATAT', 'NIID_5'), ('GCCATTTACTGAAGGG', 'NIID_5'), ('ATTAGATTATAACGTA', 'NIID_6'), ('TAAAAAGCCTCCATGA', 'NIID_6'), ('CCCCCGCTGTTTAAAA', 'NIID_7'), ('GCCTTTGCGCGCAGTC', 'NIID_7'), ('TTGACTCACCGAATAA', 'NIID_7'), ('GTGCATCCTAGTGACG', 'NIID_8'), ('CAATTCGCCGTTCCCC', 'NIID_9')]

Fit neutralization curves after applying QC

No we re-fit curves after applying all the QC:

fits_qc = neutcurve.CurveFits(
    frac_infectivity.rename(
        columns={
            "frac_infectivity_ceiling": "fraction infectivity",
            "concentration": "serum concentration",
        }
    ),
    conc_col="serum concentration",
    fracinf_col="fraction infectivity",
    virus_col="strain",
    serum_col="serum",
    replicate_col="plate_barcode",
    fixtop=curvefit_params["fixtop"],
    fixbottom=curvefit_params["fixbottom"],
    fixslope=curvefit_params["fixslope"],
)

fit_params_qc = fits_qc.fitParams(average_only=False, no_average=True)
assert len(fit_params_qc) <= len(
    fits_noqc.fitParams(average_only=False, no_average=True)
)

print(f"Assigning fits for this plate to {group}")
fit_params_qc.insert(0, "group", group)

Assigning fits for this plate to NIID

Plot all the curves that passed QC:

if fits_qc.sera:
    fig, _ = fits_qc.plotReplicates(
        attempt_shared_legend=False,
        legendfontsize=8,
        titlesize=9,
        ticksize=10,
        ncol=6,
        draw_in_bounds=True,
    )
    display_curve_fig(fig, curve_display_method)
    plt.close(fig)
else:
    print("No sera passed QC.")

Save results to files

print(f"Writing fraction infectivities to {frac_infectivity_csv}")
(
    frac_infectivity[
        [
            "serum",
            "strain",
            "plate_barcode",
            "dilution_factor",
            "frac_infectivity_raw",
            "frac_infectivity_ceiling",
        ]
    ]
    .sort_values(["serum", "plate_barcode", "dilution_factor"])
    .to_csv(frac_infectivity_csv, index=False, float_format="%.4g")
)

print(f"\nWriting fit parameters to {fits_csv}")
(
    fit_params_qc.drop(columns=["nreplicates", "ic50_str"]).to_csv(
        fits_csv, index=False, float_format="%.4g"
    )
)

print(f"\nPickling neutcurve.CurveFits object for these data to {fits_pickle}")
with open(fits_pickle, "wb") as f:
    pickle.dump(fits_qc, f)

print(f"\nWriting QC drops to {qc_drops_yaml}")


def tup_to_str(x):
    return " ".join(x) if isinstance(x, tuple) else x


qc_drops_for_yaml = {
    key: {tup_to_str(key2): val2 for key2, val2 in val.items()}
    for key, val in qc_drops.items()
}
with open(qc_drops_yaml, "w") as f:
    yaml.YAML(typ="rt").dump(qc_drops_for_yaml, f)
print("\nHere are the QC drops:\n***************************")
yaml.YAML(typ="rt").dump(qc_drops_for_yaml, sys.stdout)

Writing fraction infectivities to results/plates/plate16/frac_infectivity.csv

Writing fit parameters to results/plates/plate16/curvefits.csv

Pickling neutcurve.CurveFits object for these data to results/plates/plate16/curvefits.pickle

Writing QC drops to results/plates/plate16/qc_drops.yml

Here are the QC drops:
***************************
wells: {}
barcodes:
  AGTCCTATCCTCAAAT: min_neut_standard_frac_per_well
  CTCTTACGCTCCTACG: min_neut_standard_frac_per_well
  GATTCAGATGCCCACC: min_neut_standard_frac_per_well
barcode_wells:
  TACAAGAGAGGGGTCC A12: min_no_serum_count_per_viral_barcode_well
  CCTTTCTCAAAACATA G12: min_no_serum_count_per_viral_barcode_well
  TACAAGAGAGGGGTCC H12: min_no_serum_count_per_viral_barcode_well
  AGATCCACCCTATAGT F1: max_frac_infectivity_per_viral_barcode_well
  CACCAATCTTCGAACT F2: max_frac_infectivity_per_viral_barcode_well
  GAGGGGATACGTCACC G2: max_frac_infectivity_per_viral_barcode_well
  GCATGGAACTAACTCC D3: max_frac_infectivity_per_viral_barcode_well
  GATCACGCAGAAAAAG E3: max_frac_infectivity_per_viral_barcode_well
  AGGACTATAGTTGGCA F3: max_frac_infectivity_per_viral_barcode_well
  CACCAATCTTCGAACT F3: max_frac_infectivity_per_viral_barcode_well
  ATGGTTTTACGTCCAT H3: max_frac_infectivity_per_viral_barcode_well
  AGATCCACCCTATAGT D4: max_frac_infectivity_per_viral_barcode_well
  CGCGACACCCTTCCGG E4: max_frac_infectivity_per_viral_barcode_well
  TATTAAGAGAAGTGCG E4: max_frac_infectivity_per_viral_barcode_well
  TCGAGTTAATATGCGC E4: max_frac_infectivity_per_viral_barcode_well
  GTGGTATCAAGCCGGG G4: max_frac_infectivity_per_viral_barcode_well
  CCGGATAAATCAGAAC G4: max_frac_infectivity_per_viral_barcode_well
  GAAAGTCCCTATGATG G4: max_frac_infectivity_per_viral_barcode_well
  ATGGTTTTACGTCCAT G4: max_frac_infectivity_per_viral_barcode_well
  GCAGCGTGCCGGTCAT H4: max_frac_infectivity_per_viral_barcode_well
  CACCAATCTTCGAACT B5: max_frac_infectivity_per_viral_barcode_well
  CGCGACACCCTTCCGG E5: max_frac_infectivity_per_viral_barcode_well
  CGTGACCCCCTCCAAC E5: max_frac_infectivity_per_viral_barcode_well
  TCGCTTCAACTAAAAA E5: max_frac_infectivity_per_viral_barcode_well
  ATGGTTTTACGTCCAT E5: max_frac_infectivity_per_viral_barcode_well
  CACCTAGGATCGCACT G5: max_frac_infectivity_per_viral_barcode_well
  AGATCCACCCTATAGT F6: max_frac_infectivity_per_viral_barcode_well
  CACCAATCTTCGAACT G6: max_frac_infectivity_per_viral_barcode_well
  GCCGCTGCGGCGTGTG H6: max_frac_infectivity_per_viral_barcode_well
  CCTTTCTCAAAACATA E7: max_frac_infectivity_per_viral_barcode_well
  GACCCCTTGTAAGATG F7: max_frac_infectivity_per_viral_barcode_well
  AGGTTCAGACTCTTGC G7: max_frac_infectivity_per_viral_barcode_well
  GACCCCTTGTAAGATG G7: max_frac_infectivity_per_viral_barcode_well
  TCTCAGCTCTTAGCCG H7: max_frac_infectivity_per_viral_barcode_well
  GACCCCTTGTAAGATG E8: max_frac_infectivity_per_viral_barcode_well
  AACCGTACCGCGTTTA G8: max_frac_infectivity_per_viral_barcode_well
  CGAAACACGTCCCAGT G8: max_frac_infectivity_per_viral_barcode_well
  CCTTTCTCAAAACATA G8: max_frac_infectivity_per_viral_barcode_well
  GTGCATCCTAGTGACG G8: max_frac_infectivity_per_viral_barcode_well
  AGATCCACCCTATAGT H8: max_frac_infectivity_per_viral_barcode_well
  ATAACTGAGGGCATTG E9: max_frac_infectivity_per_viral_barcode_well
  AATGCGAGCATGTCAA E9: max_frac_infectivity_per_viral_barcode_well
  GTGCATCCTAGTGACG E9: max_frac_infectivity_per_viral_barcode_well
  TCGAGTTAATATGCGC E9: max_frac_infectivity_per_viral_barcode_well
  AATGCGAGCATGTCAA G9: max_frac_infectivity_per_viral_barcode_well
  CACTAGATGTACAGTC G9: max_frac_infectivity_per_viral_barcode_well
  GAAATCCCCAAATAAC G9: max_frac_infectivity_per_viral_barcode_well
  AGATCCACCCTATAGT G9: max_frac_infectivity_per_viral_barcode_well
  TCGAGTTAATATGCGC G9: max_frac_infectivity_per_viral_barcode_well
  CACCAATCTTCGAACT G9: max_frac_infectivity_per_viral_barcode_well
  CCTTTCTCAAAACATA G9: max_frac_infectivity_per_viral_barcode_well
  AAAGCTCTTTTCGTTC H9: max_frac_infectivity_per_viral_barcode_well
  GCATGGAACTAACTCC H9: max_frac_infectivity_per_viral_barcode_well
  TAATAAGCCAGCAAGA H9: max_frac_infectivity_per_viral_barcode_well
  CTTACAGAATACTAGA H9: max_frac_infectivity_per_viral_barcode_well
  CGACTCCACGGACGCC H9: max_frac_infectivity_per_viral_barcode_well
  GACCCCTTGTAAGATG H9: max_frac_infectivity_per_viral_barcode_well
  ATGGTTTTACGTCCAT D10: max_frac_infectivity_per_viral_barcode_well
  AATGCGAGCATGTCAA E10: max_frac_infectivity_per_viral_barcode_well
  GCCGGCGTTAGTGTCA F10: max_frac_infectivity_per_viral_barcode_well
  CCTTTCTCAAAACATA F10: max_frac_infectivity_per_viral_barcode_well
  ACAGTACGATCTACGC G10: max_frac_infectivity_per_viral_barcode_well
  TTTATATCGAGATTCA E11: max_frac_infectivity_per_viral_barcode_well
  TCGATTACTAGCCGGA G11: max_frac_infectivity_per_viral_barcode_well
barcode_serum_replicates:
  ACACGGGTTGGCTGTA NIID_1: goodness_of_fit
  AAAGACCTTTAACTCT NIID_10: goodness_of_fit
  AACCACCCCAGAGATG NIID_10: goodness_of_fit
  ATTTACTCATTATACG NIID_10: goodness_of_fit
  GTAAGCTTCATGGAGT NIID_10: goodness_of_fit
  AGACCATCGCACCCAA NIID_11: goodness_of_fit
  AGTGTTGGCTTGGTTA NIID_11: goodness_of_fit
  AGTTGGGGTCTCCCTT NIID_11: goodness_of_fit
  CCCTTTACGGATCTCT NIID_11: goodness_of_fit
  CCGCATTAGCGGGAGG NIID_11: goodness_of_fit
  CGGTCGGGACTCATCT NIID_11: goodness_of_fit
  CGTACGTATGTCCCAG NIID_11: goodness_of_fit
  CTTTTCTAGTACGCTT NIID_11: goodness_of_fit
  GAAGTGCTGCTGAAGT NIID_11: goodness_of_fit
  GCCGCTGCGGCGTGTG NIID_11: goodness_of_fit
  GTAAGCTTCATGGAGT NIID_11: goodness_of_fit
  ACCCCCGGAGCTTGGC NIID_4: goodness_of_fit
  AGATCCACCCTATAGT NIID_4: goodness_of_fit
  CTGAACTTGTCGATAT NIID_5: goodness_of_fit
  GCCATTTACTGAAGGG NIID_5: goodness_of_fit
  ATTAGATTATAACGTA NIID_6: goodness_of_fit
  TAAAAAGCCTCCATGA NIID_6: goodness_of_fit
  CCCCCGCTGTTTAAAA NIID_7: goodness_of_fit
  GCCTTTGCGCGCAGTC NIID_7: goodness_of_fit
  TTGACTCACCGAATAA NIID_7: goodness_of_fit
  GTGCATCCTAGTGACG NIID_8: goodness_of_fit
  CAATTCGCCGTTCCCC NIID_9: goodness_of_fit
serum_replicates: {}