multiple alignment of RADtags

[iwex]

I have done multiple alignment of RAD sequences. In generated bam file I can see that for many contigs aligned sequences are one nucleotide shifted in relation to the reference. I work with non-model species and as the reference the most highly represented read in a group is used.
I attach the picture of bam file in Tablet.

Do you have any idea what could cause this problem?

[tcezard]

Can you be a bit more explicit about the tool you use?
How did you make the groups you're talking about?
Did you align the reads to your reference or did an actual MSA.

[iwex]

Tcezard, thanks for your interest.
First I used blat, then mcl graph clustering, then muscle for multiple alignment. It was multiple sequence alignment. And the output of that was both sam/bam file and fasta reference sequences file.

I am doing double digest RADseq experiment based on the paper of Brandt Peterson "Double Digest RADseq: An Inexpensive method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species" (2012), PLos ONE.

And for the analyses I use scripts (rtd) mentioned in the paper and available at github (https://github.com/brantp/rtd).

Maybe someone has been using this method?

[ccortes]

Hi Iwex do you have the idea of how does de ddRADseq works I mean about the workflow?

[iwex]

Hi Ccortes,
yes I know the workflow quite well. I have already done ddRAD libraries on my own. And finaly I analysed them with Stacks as I could not solve the mentioned problem with alignment.
Do you have any questions?

[JackieBadger]

we also used stacks as the ddRAD pipeline would produce HUGE temp files.

[ccortes]

Thank you iwex, currently iḿ comparing diferent tools for implementing a pipeline for SNP calling. I read about the ddRADseq pipeline however i don't the startign point for this pipeline, could you tell how did you do the analysis.

Best regards

Hi Ccortes,
yes I know the workflow quite well. I have already done ddRAD libraries on my own. And finaly I analysed them with Stacks as I could not solve the mentioned problem with alignment.
Do you have any questions?[/QUOTE]