data

Input data

PacBio full-length variant sequencing

PacBio_runs.csv contains information on PacBio runs linking the barcodes to the mutations. It has the following columns:

• library: name of the library sequenced
• run: date of the pacbio library submission (use this date to refer to experimental notebook)
• fastq: FASTQ file from running CCS

The amplicons itself is defined in PacBio_amplicon.gb, and PacBio_feature_parse_specs.yaml specifies how to parse that with alignparse.

Illumina barcode sequencing

barcode_runs.csv has the Illumina barcode-sequencing runs used to count barcodes in different conditions. It describes the samples, which should be named as clearly as possible. It has the following columns:

• date: date experiment was performed in YYYY-MM-DD encoding.
• virus_batch: batch of virus used for the experiment.
• library: which virus library was used.
• sample_type: can be one of the following:
  • VSVG_control: entry mediated by VSVG
  • no-antibody_control: entry mediated by VEP of interest
  • antibody: encompasses sera and antibodies
• antibody: name of the antibody if this sample has sample_type of antibody
• antibody_concentration: concentration of antibody if this sample has sample_type of antibody. For sera, should be a fraction < 1 giving dilution (not a dilution factor).
• replicate: experimental replicate.
• fastq_R1: path to R1 FASTQ file, or semi-colon de-limited list of multiple FASTQs
• exclude_after_counts: set to yes if barcode run should be excluded after counting barcodes
• notes: any other notes about the sample.

The file neutralization_standard_barcodes.csv has the barcodes for the neutralization standard that is not expected to be neutralized by the sera or antibodies.

Assign sites to protein regions (domains)

reference_site_to_region.csv assigns sites in the protein to a region (protein domain).

Configuration for polyclonal analysis

polyclonal_config.yaml specifies how the analysis with polyclonal is done. For each antibody listed in barcode_runs.csv, specify:

• max_epitopes: the maximum number of epitopes to test. The fitting keeps testing more epitopes up to this max until additional epitopes have no additional value.
• n_bootstrap_samples: number of bootstrap samples to use.
• min_epitope_activity_to_include: keep adding epitopes until activity <= this.
• fit_kwargs: keyword arguments passed to Polyclonal.fit
• plot_kwargs: keyword arguments passed to Polyclonal.plot_mut_escape.

Validation neutralization assays

validation_IC50s.csv contains mutations for which we want to calculate IC50s using the polyclonal fits and compare to measured values in validation experiments. Required columns are "antibody", "aa_substitutions", "measured IC50". If multiple mutations, they should be space delimited in "aa_substitutions" columns.