Good morning,
I'm a newbie of this place and bioinformatic too, sorry in advantage if there would be any mistake in my questions.
I'm trying to analyze NGS results from amplicons sequencing (Ion torrent, coming form a lentiviral infection with pCDH plasmid containing sequences of pre-miRNAs). The samples were obtained by PCR amplification at exponential phase with pCDH specific primers of the region of interest I wanted to amplify.
The median of the reads obtained by NGS is 140-150n. What I've done up to now is cut out the primers from the BAM file (the sequence is already know) and align the samples to a reference genome (hg38) with bowtie2 tool in galaxy.
The SAM files obtained were uploaded to SeqMONK tool. Visualizing the aligned reads I see a lot of artifacts (or what I think they are). The idea is to count the "differential representation" (enrichment?) of the pre-miRNAs in two different conditions.
![Seqmonk][1]: https://ibb.co/1ZpmsyR
My questions are:
- should it be better to align the reads to a custom reference genome with only the features present in my infection, it means whit only the pre-miRNA contained in the amplicons obtained by PCR?
- what I see are effectively artifacts or part of the genome obtained by amplification becose they trapped the primes (the primers were basted).
- It is conceptually right to use DESeq2 to analyze this pre-miRNAs overrepresentation in my two conditions?
thank you for your help, G.
