Seqman Leaves most of the reads unaligned

[Mansequencer]

Hi All,
The job at hand it to align 17 million 50 bp illumina reads onto a reference scaffold through Seqman Ngen. The results from the alignment show that approx. 16 million reads remain unaligned when ran with default parameters. Even tweaking the parameters (minmatch percentage=80 and match size=17) improved the alignment but only by a few percentage points.
Alignment done with same samples with Newbler showed 97% reads mapping onto the reference.
I dont have a license for Newbler and I am trying to make Seqman Ngen work. Any suggestions to improve the assembly.
Thanks,

[nickloman]

Hi All,
The job at hand it to align 17 million 50 bp illumina reads onto a reference scaffold through Seqman Ngen. The results from the alignment show that approx. 16 million reads remain unaligned when ran with default parameters. Even tweaking the parameters (minmatch percentage=80 and match size=17) improved the alignment but only by a few percentage points.
Alignment done with same samples with Newbler showed 97% reads mapping onto the reference.
I dont have a license for Newbler and I am trying to make Seqman Ngen work. Any suggestions to improve the assembly.
Thanks,

You aligned Illumina reads with Newbler? That doesn't sound right.

I haven't used Ngen - have you tried Bowtie as a comparator?

[Mansequencer]

Hi nickloman,
Thanks for your response. Yes, I was surprised too but the Newbler analysis was done by someone else. It can however complete the job but accuracy and quality is compromised if Illumina 50 bp seq. are used. Bowtie is for linux and I have a windows server. I do have Bowtie and SOAP2 in my mind and will get to it if I can find a linux server.
Thanks,

[SoftGenetics]

You might wish to try a trial of NextGene its assembler is quite robust

[Mansequencer]

Sure. I will give it a shot.
Thanks,

[bkanders]

I would recommend contacting the Next Gen application scientist at DNASTAR. Every project is different, and parameters may need to be changed accordingly. Are you removing linkers, setting the correct read type, trimming by quality, performing an iterative assembly, etc?