Barcode: Design and validate NGS barcodes¶

https://readthedocs.org/projects/barcode/badge/?version=latest

Barcode is a program for the design and validation of sets of sequencing barcodes.

Please see ReadTheDocs for the latest documentation.

Introduction¶

Barcodes are used in NGS to tag samples before pooling. After sequencing, these barcodes are used to demultiplex the data, thereby assigning the reads to the originating sample.

The key aspect of a good set of barcodes is robustness against read errors. One read error should not be able to transform one barcode into another. This requirement can be met by selecting barcodes in such a way that the edit distance between any pair of barcodes is larger than one. An additional desired property is the ability to correct read errors. This can be done by increasing the minimal edit distance between barcodes to at least three. If one read error occurs, the sequenced barcode will have a distance of one to the original barcode and a minimum distance of two to any of the other barcodes. If the read error is high, the minimum edit distance should be increased to a higher (odd) number.

For some sequencers it is important that mononucleotide stretches in barcodes are below a minimum length. An additional filter can be used to remove these barcodes.

Installation¶

The software is distributed via PyPI, it can be installed with pip:

pip install barcode

From source¶

The source is hosted on GitHub, to install the latest development version, use the following commands.

git clone https://github.com/jfjlaros/barcode.git
cd barcode
pip install .

Usage¶

The barcode program has two subcommands; one for the creation of a set of barcodes and one for the validation of an existing set of barcodes.

To make a set of barcodes and write this set to a file named barcodes.txt, use the following command:

barcode make barcodes.txt

barcodes.txt will now contain a list of barcodes that all have length 8, and no barcode will contain a mononucleotide stretch longer than 2.

The length of the barcodes can be controlled with the -l parameter, the minimum edit distance is controlled with the -d option and the maximum mononucleotide stretch length can be set with the -s option. So if we want to make a list of barcodes of length 10, a minimum edit distance of 5 (allowing for the correction of 2 read errors) and a maximum mononucleotide stretch of 1, we use the following command:

barcode make -d 5 -l 10 -s 1 barcodes.txt

To verify a list of existing barcodes, use the command:

barcode test barcode.txt

This will check the distance between any pair of barcodes and will tell you how many barcodes violate the distance constraint. Again, the minimum edit distance can be set with the -d parameter.

Additionally, a good set of barcodes can be extracted by providing an output file via the -o option:

barcode test -o good_barcodes.txt barcodes.txt

Command Line Interface¶

Design and test NGS barcodes.

usage: barcode [-h] [-v] {make,test} ...

Positional Arguments¶

subcommand: Possible choices: make, test

Named Arguments¶

-v: show program’s version number and exit

Sub-commands¶

make¶

Make a set of barcodes, filter them for mononucleotide stretches and for: distances with other barcodes.

barcode make [-h] [-d DISTANCE] [-H] [-l LENGTH] [-s STRETCH] OUTPUT

Positional Arguments¶

OUTPUT: output file

Named Arguments¶

-d

minimum distance between the barcodes (int default=3)

Default: 3

-H

use Hamming distance

Default: False

-l

lenght of the barcodes (int default=8)

Default: 8

-s

maximum mononucleotide stretch length (int default=2)

Default: 2

test¶

Test a set of barcodes.

barcode test [-h] [-d DISTANCE] [-H] [-o OUTPUT] INPUT

Positional Arguments¶

INPUT: input file

Named Arguments¶

-d

minimum distance between the barcodes (int default=3)

Default: 3

-H

use Hamming distance

Default: False

-o

list of good barcodes

Copyright (c) Jeroen F.J. Laros <J.F.J.Laros@lumc.nl>

Library¶

Barcode design via the library is done in three steps. First obtain the full set of permutations with the all_barcodes function:

>>> from barcode import all_barcodes, filter_distance, filter_stretches
>>>
>>> # Generate all barcodes of length 2.
>>> all_barcodes(2)
['AA', 'AC', 'AG', 'AT', 'CA', 'CC', 'CG', 'CT', 'GA', 'GC', 'GG', 'GT', 'TA',
'TC', 'TG', 'TT']

The resulting list can be filtered with the filter_distance and filter_stretches functions:

>>> # Filter all barcodes of length 3 for a minimal edit distance of 3.
>>> filter_distance(all_barcodes(3), 3)
['AAA', 'CCC', 'GGG', 'TTT']
>>>
>>> # Filter all barcodes of lenght 2 for mononucleotide stretches of length
>>> # longer than 1.
>>> filter_stretches(all_barcodes(2), 1)
['AC', 'AG', 'AT', 'CA', 'CG', 'CT', 'GA', 'GC', 'GT', 'TA', 'TC', 'TG']

For the best result, apply the filter_stretches function before applying the filter_distance function:

>>> # Make a set of barcodes of length 3, having no mononucleotide stretches
>>> # and a minimum edit distance of 3.
>>> filter_distance(filter_stretches(all_barcodes(3), 1), 3)
['ACA', 'CGC', 'GAG']

API documentation¶

barcode.barcode.all_barcodes(length)¶

Generate all possible barcodes of a certain length.

Parameters: length (int) – Lenth of the barcodes.
Returns list: List of barcodes.

barcode.barcode.filter_distance(barcodes, min_dist, distance=<function distance>)¶

Filter a list of barcodes for distance to other barcodes.

Parameters

barcodes (list) – List of barcodes.
min_dist (int) – Minimum distance between the barcodes.
distance (function) – Distance function.

Returns list

List of barcodes filtered for distance to other barcodes.

barcode.barcode.filter_stretches(barcodes, max_stretch)¶

Filter a list of barcodes for mononucleotide stretches.

Parameters

barcodes (list) – List of barcodes.
max_stretch (int) – Maximum mononucleotide stretch length.

Returns list

List of barcodes filtered for mononucleotide stretches.

Contributors¶

Jeroen F.J. Laros <J.F.J.Laros@lumc.nl> (Original author, maintainer)

Find out who contributed:

git shortlog -s -e