Partial amplicon region missing from mapping data

Question

[lakhujanivijay]

I have a 1 kb amplicon sequenced on Ion Torrent platform that belongs to a virus. Statistics are given below

file            virus_genome.fa     data.fastq
format          FASTA               FASTQ
type            DNA                 DNA
num_seqs        1                   60231
sum_len         9181                9416001
min_len         9181                25
avg_len         9181                156.3
max_len         9181                344

I have mapped those reads on to the virus genome using bwa with 88% mapping, but those reads only mapped to around 600-700 bps out of the entire genome. Look at below image from the IGV viewer.

I am wondering why there is no mapping covering the entire 1 kb amplicon; i.e. I am still missing ~ 400bps from my 1kb amplicon. Is it something to do with the wetlab or is it about the mapping (soft clipped bases somewhere) ?

Any help is much appreciated!

Answer 1

[swbarnes2]

Your reads diverge a lot from your reference. At first glance, I'd say you didn't sequence what you thought you sequenced. I'd assemble them and blast that against nr, and see what you really have,

[lakhujanivijay]

That's exactly what I did and unfortunately the assembly is very much fragmented. How can I check read quality outside torrent suite? Would fastqc be suffice for that ?

[swbarnes2]

Read quality is probably not your problem. That region is small, make the consensus manually

[lakhujanivijay]

Any idea how do I proceed about that ?