There are a few key considerations when analyzing sequencing data generated from the xGen HS EGFR Pathway Amplicon Panel with unique molecular identifiers (UMIs).
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). Also, before aligning the reads, make sure that the 10 bp UMI (which contains random bases) has been trimmed off from 5’ of Read 2.
In addition, check that adapter trimming is enabled while setting up the sequencing run. Alternatively, adapter trimming may be performed bioinformatically before 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.
For more advice, you may wish to contact our Scientific Application Supports team.
Note: A target BED file is provided with purchase of the xGen HS EGFR Pathway Amplicon Panel or the xGen Custom Amplicon Panel.