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ECBC

Subtype assignment and primer trimming for antibody sequences with Error Correcting BarCodes (ECBC)

Experimental Setup

ECBC IgG Reads

  • R1: fwd 4N, leader, variable
  • R2: rev subtype determination, constant, variable
  • I1: ECBC (12)
  • I2: sample index

ECBC klMA Reads

  • R1: fwd 4N, leader, variable
  • R2: rev ECBC (12), spacer (4), constant, variable
  • I1: LC index TAAGGCGAGAGC (12)
  • I2: sample index

Workflow

IgG_klMA_ECBC.py

  1. split into IgG vs. klMA
  2. for LC do klMA discrimination
  3. for HC determine IgG subtypes
  4. write ECBC in front of R1
  5. pandaseq R1 und R2

Usage

IgG_klMA_ECBC_V2.py NAME_L001_R1_001.FASTQ[.GZ]

###Helper Scripts seq_count.sh counts fasta and fastq sequences

batch.ch to run multiple samples

imgt_combine.sh to combine two IMGT output files from the same sample

Primer

  • primer_fwd_H.fasta heavy chain forward primers
  • primer_fwd_k.fasta kappa forward primers
  • primer_fwd_l.fasta lambda forward primers

Analysis ECBC Ab sequences

  1. run IgG_klMA_ECBC_V2.py
  2. upload sequences to IMGT (split big files into files with 1000000 sequences each)
  3. download IMGT results, combine files which had been split before
  4. run annotate_first.py