barcodecop 0.2

Enforce exact barcode matches in demultiplexed MiSeq reads

Enforce exact barcode matches in demultiplexed MiSeq reads

The onboard software used for demultiplexing on the Illumina MiSeq cannot be configured to enforce exact barcode matches. As a result, a minority of reads (up to about 5% in my tests) are assigned to a specimen on the basis of a partial barcode match. It turns out that some fraction of these less-than-exact matches are mis-assigned from other specimens (as of course are some smaller fraction of the exact matches, but we can’t identify these as easily). This mis-assignment is a problem for ultra sensitive assays that attempt to draw conclusions from the presence of very low prevalence reads.

This package provides the barcodecop command that uses the index reads to determine the most prevalent barcode sequence and filter reads from an accompanying fastq file.

Command line arguments:

usage: barcodecop [-h] [-f file.fastq[.bz2|.gz]] [-o OUTFILE] [--snifflimit N]
                  [--head N] [--min-pct-assignment PERCENT] [--invert] [-c]
                  [-q] [-V]
                  file.fastq[.bz2|.gz] [file.fastq[.bz2|.gz] ...]

Filter fastq files, limiting to exact barcode matches.

Input and output files may be compressed as indicated by a .bz2 or .gz
suffix.

positional arguments:
  file.fastq[.bz2|.gz]  one or two files containing index reads in fastq
                        format

optional arguments:
  -h, --help            show this help message and exit
  -f file.fastq[.bz2|.gz], --fastq file.fastq[.bz2|.gz]
                        reads to filter in fastq format
  -o OUTFILE, --outfile OUTFILE
                        output fastq
  --snifflimit N        read no more than N records from the index file
                        [10000]
  --head N              limit the output file to N records
  --min-pct-assignment PERCENT
                        raise error unless the most common barcode represents
                        at least PERCENT of the total [90.0]
  --invert              include only sequences *not* matching the most common
                        barcode
  -c, --show-counts     tabulate barcode counts and exit
  -q, --quiet           minimize messages to stderr
  -V, --version         Print the version number and exit

Both single and dual-indexing are supported. For example:

barcodecop input_I1.fastq --fastq input_R1.fastq -o output_R1.fastq

Or, using a dual index:

barcodecop input_I1.fastq input_I2.fastq --fastq input_R1.fastq -o output_R1.fastq

Installation

Install from PyPi using pip:

pip install barcodecop

Or install directly from the git repository:

pip install git+https://github.com/nhoffman/barcodecop.git

Testing

Run all tests like this:

python setup.py test