Alignment

[leticiaraposo]

Hello everybody,

I'm new in NGS area and I would like to solve some questions.
One of them is:

I have many NGS sequences in a BAM format:

For example, for a same sequence, I have:

4M1D1M1D4M2I2M114S

CTTCCCCCACCACCGTTGGCACAGCGCCCCCGGGAACACCCTCCCACCACCACCGTCGGCACAGCGCCCCGGGGAGCACCCCAGCCCAGCTGCACCAGGGCTCTCTGAAGGAGGTGGTGGTCCGGTT

CTTC-C-CCCAAC

I don't understand why a great part of sequence was missed when it was converted CIGAR to aligned sequence. Can anyone help me?

Other question is: which sequence I use to convert it into an amino acid sequence?

CTTCCCCCACCACCGTTGGCACAGCGCCCCCGGGAACACCCTCCCACCACCACCGTCGGCACAGCGCCCCGGGGAGCACCCCAGCCCAGCTGCACCAGGGCTCTCTGAAGGAGGTGGTGGTCCGGTT or CTTC-C-CCCAAC?

In fact, I want to compare amino acids mutations in relation to the reference sequence.

Sorry for my english and thanks so much for the answers.

[GenoMax]

A large part of your read was soft-clipped by the aligner you used. That part appears to belong to human chr17.

What kind of experiment is this data from? You may have to consider all frames if this is plain re-sequencing.

[leticiaraposo]

Firstly, thank you for your reply.
This is a HIV sequence and it was aligned with TMAP aligner using HIV HXB2 reference.
I would like to check which HIV quasispecies are present in a patient using NGS.
The problem is that I'm mathematician and I don't have a good basis in this area of bioinformatics.
Do you have some good references to show me?

Thanks!

[GenoMax]

Firstly, thank you for your reply.
This is a HIV sequence and it was aligned with TMAP aligner using HIV HXB2 reference.
I would like to check which HIV quasispecies are present in a patient using NGS.
The problem is that I'm mathematician and I don't have a good basis in this area of bioinformatics.
Do you have some good references to show me?

Thanks!

If I just take the read you posted and use it to blast at NCBI then I am getting the following hit. So this particular read has a good match on human Chromosome 17 clone RP11-388C12 GenBank accession AC024361.21 (it may be related to HIV somehow).

You will have to consider the possibility that there may be other human DNA in your sample (e.g. this read may be an example of that).

Query  1      CTTCCCCCACCACCGTTGGCACAGCGCCCCCGGGAACACCCTCCCACCACCACCGTCGGC  60
              ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  15201  CTTCCCCCACCACCGTTGGCACAGCGCCCCCGGGAACACCCTCCCACCACCACCGTCGGC  15142

Query  61     ACAGCGCCCCGGGGAGCACCCC-AG-CCCAGCTGCACCAGGGCT-CTCTGAAGGAGGTGG  117
              |||||||||||||||||||||| || |||||||||||||||||| |||||||||||||||
Sbjct  15141  ACAGCGCCCCGGGGAGCACCCCCAGCCCCAGCTGCACCAGGGCTTCTCTGAAGGAGGTGG  15082

Query  118    TGGTCCGGTT  127
              ||||||||||
Sbjct  15081  TGGTCCGGTT  15072

Here is a not-so-comprehensive guide for a non-biologist for NGS sequence data alignments: http://en.wikibooks.org/wiki/Next_Ge...S%29/Alignment