How do I analyze xGen™ HS EGFR Pathway Amplicon Panel data?
There are a few key considerations when analyzing sequencing data generated from the xGen HS EGFR Pathway Amplicon Panel with unique molecular identifiers (UMI).
The first 10 bases in front of Read 2 constitute a UMI. Therefore we recommend using Trimmomatic to trim (CROP) these first 10 bases from Read 2 to make an MID/UMI fastq file for use with the MID pipeline from the fgbio package (Fulcrum Genomics; https://github.com/fulcrumgenomics/fgbio). In addition, prior to aligning the reads, make sure that the 10 bp UMI (which contains random bases) has been trimmed off from 5’ of Read 2.
Further, please check that adapter trimming is enabled while setting up the sequencing run. Alternatively, adapter trimming may be performed bioinformatically prior to analysis.
xGen Custom Amplicon Panels are designed with overlapping amplicons to allow for contiguous regions of coverage in a single-tube format. Therefore, synthetic primer sequences will be encountered both at the beginning and end of some reads, which must be trimmed during analysis. This can be done using a publicly available tool called Primerclip. Review Primerclip-A Tool for Trimming Primer Sequences Application Note for more information.
If additional advice is needed, contact IDT's Scientific Application Support team at firstname.lastname@example.org
A target BED file is provided with purchase of the xGen HS EGFR Pathway Amplicon Panel or xGen Custom Amplicon Panel.