#allprep Tool for preprocessing and demultiplexing of sequencing data.
Barcode Preparation Toolbox README
DISCLAIMER: Meric Lieberman, 2016 This work is the property of UC Davis Genome Center - Comai Lab This is shared under a Creative Commons BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/
Use at your own risk. We cannot provide support. All information obtained/inferred with these scripts is without any implied warranty of fitness for any purpose or use whatsoever.
SUMMARY: The scripts included are for use in preparation of raw illumina reads for further analysis. Traditionally, we do all prep work with a single script called "Allprep", that does barcode check and match, 'N' filtering, primary and secondary adapter contamination, quality conversion, quality trimming, length trimming, and library separation.
However, if parts of the preparation process needs to be performed independently, we also provide smaller scripts to do the process modularly.
Please note that:
- all of the scripts have a description of what they do, input parameters, and running directions at the beginning of each program.
- all scripts use command line parameters as input and all scripts can be run with ./"programname" provided Python 2.4+ is installed on your system.
- these scripts can be used with paired end or single ended data, and some can be run on data without barcodes.
CONTENTS:
This .zip includes:
allPrep-13.py - Single script to do all processing as detailed above. README-barcode-file.txt - An explanation of the barcode file format sample-barcode-file.txt - An example barcode / index file
interleaveSwitcher.py - This script will interleave two files or uninterleave a file. This is only applicable to paired-end reads. "Interleaved" means that the two ends of a read are placed together in the final file. View parameters in script for running directions.
SCRIPT DETAILS: The following information can be found at the top of every script, after disclaimer and library import.
allprep-13.py
Usage: For all modes the run command looks like this, with [...] indicating files needed by specific read type, and {....} indication optional parameters allprep-13.py -b barcode-file.txt -f forward-read-file.fq [-r reverse-read-file.fq] [-i index1-file.fq] [-I index2-file.fq] {-m} {-E} {-n} {-q} {-d}
For a quick view of parameters form the command line, simply use the -h for help. allprep-13.py -h
This program that takes a barcode file and splits the lane sequence.txt files into specified library.txt (lib#.txt) files, does barcode check and match, 'N' filtering, primary and secondary adapter contamination, quality conversion, quality trimming (mean quality of 20 over 5 base window), length trimming (default 35), and library separation.
Input: This script takes a barcode file as specified in the sample sheet in the README, as well as the forward read file, and optionally the reverse, index and secondary index These files are loaded with -b, -f, -r, -i, and -I respectively. There are also five additional operating options: -m for mismatch mode, this allows a 1 bp mismatch between the index / barcode and the best matching barcode. -E for error reads mode, outputs the rejected reads to a file -n for N allowed mode, this turns off the check that rejects a read if there are any 'N' nucleotides in the sequence -N for N reads, trim at the N instead of rejecting outrihgt NOTE: THIS DRAMATICALLY EXTENDS RUNTIME -q to convert a file using Illumina 1.5 qualities to Sanger/Illumina1.8 (standard) -D for only demultiplexing mode, this only splits the reads by barcode and does no additional trimming or checks -M use this to change the defualt minimum read length post filtering
This program can take a file with most combinations of barcode/indexing. The possibilities are:
- single ended, barcoded
- pair ended, barcoded
- single ended, one index
- single ended, two index
- pair ended, one index
- pair ended, two index
Please see the README-barcode-file.txt and sample-barcode-file.txt for help in creating the barcode file for your dataset.
interleaveSwitcher.py
Usage: programName.py -f forwardFile.fq -r reverseFile.fq -o outFileName.fq OR_ Usage: programName.py -f interleavedFile
This program has two operating modes, defined by hoiw many files are provided.
Interleave mode, two files are given. This will take two read files of equal length and interleave them. Input Parameters: forwardFile.fq = original file input, Sanger or illumina file forward side reverseFile = original file input, Sanger or illumina reverse side outFileName.fq = interleaved output file name/location
Uninterleave mode, only one input file given. This will take one interleaved paired read file and split them into two paired read files. The -1.fq and -2.fq result files will be forward and backward respectively.
Input Parameters: interleavedFile.fq = original file input, Sanger or illumina interleaved paired reads
use interleaveSwitcher.py -h to see help for command line parameters