Correcting sequence reads based on ORF disruption from reference sequence.

Question

[Daniel]

I have a set of 454 metagenomic (amplicon) reads from which I am attempting to extract a particular taxon group.

I have done the following:

• Select reference sequences for key taxa family (NCBI and some of our own).
• Create reference alignment using muscle.
• Align NGS query sequences to reference alignment using pynast, keeping those sequences which align closely and discarding non related taxa.

I now want to try and correct the extracted sequences based on the ORF of the functional gene which we are using (rbcl). I can see a homopolymer which is causing an insertion and others errors, but is there a way to correct the sequences?

Thanks

Answer 1

[Daniel]

I have found the RDP tool 'FrameBot' to be quite good in this respect. Takes a bit of tweaking and it seems unwilling to remove some insertions but it's the best I've come across so far

https://github.com/rdpstaff/RDPTools

RDP FrameBot is a frameshift correction and nearest neighbor classification tool for use with high-throughput amplicon sequencing. It uses a dynamic programming algorithm to align each query DNA sequence against a set of target protein sequences, produces frameshift-corrected protein and DNA sequences and an optimal global or local protein alignment. It also helps filter out non-target reads. The online version of FrameBot is available on http://fungene.cme.msu.edu/FunGenePipeline. Read the quick tutorial at http://rdp.cme.msu.edu/tutorials/framebot/RDPtutorial_FRAMEBOT.html before you start.