Why do all of my PCR reads not map to the reference sequence?

Question

[Saran]

Hello,

I was given fastq files of 150 bp illumina sequenced reads after PCR was performed on the region of interest and I was given a reference sequence of the gene. I used BWA mem to align my sequences to the reference and only got 60% mapped. I was wondering if I am using the correct algorithm for PCR produced reads and some explanation as to what could cause not all of them to map? I did not adapter trim because BWA does soft-clipping and I know some reads did sequence into the adapters because they were short. Could someone else explain as to why not all would map? Could this be PCR/sequencing error or am I doing something wrong?

Thank You

Answer 1

[GenoMax]

Did you check to see if there are phiX sequences in your data? Often times phiX is spiked in to normalize the base distribution when sequencing a low nucleotide diversity sample (amplicons would qualify for this). Generally phiX data is removed during demultiplexing but if your samples were not multiplexed phiX reads may still be there. If that is not the case grab a few reads (convert them to fasta format) that do not map and then blast them at NCBI to find out what they are.