I am testing a cencer diagnostic panel which amplifies some of the cancer "Hot spots". Manufacturer did not provide the bed file for the targets. Primer sequence has been removed by UNG and nuclease and ends of the fragments all different.
I am thinking about determine the target for the amplicon by assembling/aligning the reads. What will be the most efficient way to do this? Can I use RNAseq assembler, like cufflinks or trinity? Seems like they are overkill for this purpose though.
Thanks in advance!
1 answer
So my first piece of advice is to contact the manufacturer, they should provide you a BED file of targets, or some other similar format. If the panel is meant to be run with an Illumina machine in particular (MiSeq or NextSeq) it will have a manifest file of amplicons as it is required for their onboard workflow.
If that doesn't work (or you're impatient) I would first be a little suspicious of that manufacturer, it isn't a good business practice and doesn't speak well of them. But you can easily figure out the targets yourself. You shouldn't need an RNA Assembler, simple mapping of your reads to the reference genome will suffice. You can then use a variety of tools out there (Bedtools, etc) to scan the genome for where your reads are piled up. It will be pretty obvious. Depending on the size of the panel it is usually only a few hundred to maybe 1000 (for the bigger comprehensive panels of 100+ genes) amplicons
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