finding polymorphisms by large sequence alignments

[Alex Clop]

Hello all

I just registered to SEQanswers after a friends recommendation. I work in molecular genomics since 1996, starting in livestock genomics in Barcelona and currently working in the genetics of human diseases. I am currently analysing three overlapping human BACs (between 150 and 180 kb size each and spanning a genomic region of about 340 kb) that have been sequenced with 454 tech (done abroad). The files we have correspond to i) the reads, ii) the assembled reads in contigs, and iii) the ordered contigs (by paired end tag sequencing) into scaffolds. The file types are .fna (fasta), .qual (Phred equivalent quality scores), .tsv (tab delimited file with consensus position-by-position base and flow signal info), sff (input file used in the assembly) and .ace (to be viewed by other viewer programes).

I would like now to align these consensus sequences (all the scaffolds) to the corresponding region on the human reference genome and to other sequences with the aim to find the existing polymorphisms. From the alignment, I will extract a variation table (containing information for each polymorphism on its position, the encompassing sequence, and each allele in each sequence) as an output.

However, I haven't seen any software that fits our requirements (export a list of polymorphisms between compared large sequences). I used several alignment engines as Geniaous, BioEdit, Clustalw, but, as expected, neither the computer nor the alignment tool are powerful enough to do such analysis.

I see three different options to proceed:

1) We can either write our own programe and run it in our computers. Is that the best / only way to proceed?

2) We can download an existing programme and run it in our computers. Can I freely download a programe to do that in our servers?

3) We can use online software where we can upload our sequences and run the analysis. Is there any specific website to do thaty?

In case we decide options 2) or 3), does anyone know about any programe that would align (both pairwise and / or multi-alignment) such large sequences and would also give a list of the polymorphisms.

Thanks in advance

Kindest regards

Alex Clop

Molecular and Medical Genetics
9th Floor Guy's Tower
KCL
St Thomas' St
London SE1 9RT
UK

Tel.: +44(0)20 7188 9505
Fax: +44(0)20 7188 8050
e-mail: [email protected]

[apfejes]

I think MAQ fits your list of requirements, thought you may need to do some file conversion. I normally work with Illumina data, rather than 454, so I've never tried it with the file types you've listed above.

MAQ can be found at http://maq.sourceforge.net/

[bioinfosm]

MAQ might not handle read lengths above 60bp. So probably tools from MarthLab -http://bioinformatics.bc.edu/marthlab/Main_Page - will work best

Also, 454 has its own software newbler or something that should help. It gives a list of SNP calls as well - HCDiffs.txt

[apfejes]

Thanks for pointing that out. I had forgotten about the 60bp limit in MAQ.

[Alex Clop]

re: finding polymorphisms by large sequence alignments

Thanks for your replies.

Yes, there is Newbler which is supplied by Roche with the 454 machine. We do not have this facility here so don't have access to the software. We are thinking about developpin the tool ourselves, but honestly, I am not a bioinformatician and do not know how easy it is.

[colindaven]

Hi Alex

Before you start developing I would look at Mummer. This seems to do at least part of what you need.

http://mummer.sourceforge.net/manual/#snpdetection

I don t know if it will give you enough information about polymorphisms but perhaps you can use it as a starting point.

Cheers
Colin

[Alex Clop]

Dear Colin

Thanks very much for the info. I will have a look now.

I am currently testing the CLC Genomics Workbench from CLC Bio. It seems quite good up to now, but I am still testing it.

Best regards

Alex

[glacerda]

If possible, ask your sequencing provider to run GS Reference mapper with your data. If it is not posible try nucmer+show-snps from the Mummer package

[Josliu]

Alex,

Please use NextGene from Softgenetics to detect SNP and link to NCBI dbSNP database. I will supply a demo copy for you to use.
Josliu