MiSEQ_testbarcodes

Script for testing false/true positive reads in a MiSEQ run with custom barcodes. Used for assessing cross talk and/or sample bleed over during extraction. Includes both custom barcodes and Illumina Nextera barcodes.

Note: Probably only works on our servers in Center for Microbial Communities, Aalborg University, but feel free to adapt!

How to run

• Load R and bcl2fastq2 modules (use module spider to search for the exact names), or otherwise make sure R and bcl2fastq are available somewhere in $PATH.
• The script will install the required R packages (ggplot2 and data.table), but if it fails you must manually do that first.
• Run the script directly from GitHub or download script.sh, fxcurl -fSsl https://raw.githubusercontent.com/cmc-aau/MiSEQ_testbarcodes/master/script.sh | bash -s "/space/HiSeqUser/tempBackup/MiSeqBackup/RUNID"

What it does

• Writes out a complete SampleSheet with all nextera+custom V13 forward and reverse barcodes (including those not necessarily barcoded in the pooled library)
• Runs bcl2fastq with the complete sample sheet
• Counts ALL reads per fastq file (per barcode combination)
• Compares the original SampleSheet used for the run with the complete SampleSheet to figure out false/true positive reads
• Generates plots per barcode scheme (nextera+bv13fr) which shows the number of reads and colors by TP/FP