Novoalign: No pairs found with simNGS data

[oiiio]

I generated some paired end data using simNGS and simLibrary, as they seem to be very good for illumina HiSeq.

My problem is that novoalign does not recognize the pairs, and returns "No pairs found". It is strange because when mapping my real illumina data, it performs excellently. Any suggestions would be very helpful.

Before suggestions to use different mappers are made, I already tried BWA (which worked fine) and Stampy (same problem as novoalign). Are there known issues with simNGS and mapper compatibility?

[zee]

That is quite odd.

Are you using FASTQ simulated reads. Novoalign needs to have read1 and read2 of a pair in separate files.
Also check the coding of your intensities and use the correct coding for Illumina/Sanger FASTQ.

[sparks]

Hi,

Novoalign and Stampy both expect reads of a pair to be orientated so that one reads is on +ve strand, the other on -ve strand with 3' ends of reads towards the centre.

5---------------->3 3<-------------------5

This is how paired reads come off the Illumina. BWA doesn't apply this rule and will make proper pairs from any two adjacent mappings and is one of te reasons Novoalign and Stampy are more accurate than BWA.

My guess is that NGSsim isn't producing reads in the correct orientation.
The other problem could be the insert size produced, Novoalign and Stampy both default to around 250bp with std dev of 50bp, if NGSsim produced pairs with mean of much larger than this, say >600, then it's unlikely Stampy or Novoalign would map any pairs.
Colin

[dumps]

Hi,

be aware that the --paired option of simLibrary DEACTIVATES generation of paired end reads - Don't ask me why. But maybe your problem is that simple...

[Richard Finney]

Please give us an example of the first 12 lines of each fastq pair file.

Please note the " --hdrhd off " command line parameter available with novoalign. I think this turns off checking that names in pairs match.

Do make sure you have matching number of pair elements in fastq1 and fastq2 files.