Illumina fragment length distribution

delphi_ote · 05-04-2011, 09:51 PM

I'm trying to characterize the fragment length distribution of some Illumina data from the 1000 Genomes Project.

To do this, I downloaded the supplied BAM files (reads aligned with MAQ) for the mtDNA of the CEU trio daughter (to make sure rearrangements wouldn't be a problem.) Then I checked the distance between the two farthest ends of the mapped pairs.

I thought this would be fairly straightforward, but the mean and median aren't even close to the fragment size listed for some of the libraries. They're much lower than the listed fragment size. The distributions aren't even remotely normal, either.

For example, SRR001139 is supposed to have a fragment length of 676 with mate paired reads of size 47. The mean fragment length I estimate, however, is 234 with a standard deviation of 148.

Here is a histogram of the length distribution:

Is expected? Or am I making a mistake somewhere along the way in my reasoning? Does anyone know any references about the fragment lengths that result from Solexa sequencing?

Any help or suggestions would be much appreciated.

delphi_ote · 05-04-2011, 10:22 PM

Sorry. I said "fragment size" here somewhat carelessly. I really mean "insert size".

jnfass · 05-18-2012, 01:25 AM

randomly came across this - kinda sad that you've had no answers - doesn't bode well for the community.

In any case, could this have been coming from contaminated "mate-pair" libraries (from large circularized fragments that are then fragmented themselves - fragments that contain the circ. point lead to outward facing read pairs that map R/F at long distances, while fragments that don't contain the circ. point are F/R pairs that map at short distances. I.e. all "mate-pair" libraries are contaminated to a certain extent with shorter-range "paired-end" reads. Now, given the opposite orientations, normally one or the other set won't map, but I think MAQ was able to align both, IIRC.

HTH.
~J

rnaseek · 05-18-2012, 02:59 AM

Does Illumina library's fragment length of 676 include adapter sequences? How big were the adaptor sequences?

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05-04-2011, 09:51 PM	#1
delphi_ote Junior Member Location: Champaign, IL Join Date: Oct 2010 Posts: 9	Illumina fragment length distribution I'm trying to characterize the fragment length distribution of some Illumina data from the 1000 Genomes Project. To do this, I downloaded the supplied BAM files (reads aligned with MAQ) for the mtDNA of the CEU trio daughter (to make sure rearrangements wouldn't be a problem.) Then I checked the distance between the two farthest ends of the mapped pairs. I thought this would be fairly straightforward, but the mean and median aren't even close to the fragment size listed for some of the libraries. They're much lower than the listed fragment size. The distributions aren't even remotely normal, either. For example, SRR001139 is supposed to have a fragment length of 676 with mate paired reads of size 47. The mean fragment length I estimate, however, is 234 with a standard deviation of 148. Here is a histogram of the length distribution: Is expected? Or am I making a mistake somewhere along the way in my reasoning? Does anyone know any references about the fragment lengths that result from Solexa sequencing? Any help or suggestions would be much appreciated.

05-04-2011, 10:22 PM	#2
delphi_ote Junior Member Location: Champaign, IL Join Date: Oct 2010 Posts: 9	Sorry. I said "fragment size" here somewhat carelessly. I really mean "insert size".

05-18-2012, 01:25 AM	#3
jnfass Member Location: Davis, CA Join Date: Aug 2008 Posts: 88	randomly came across this - kinda sad that you've had no answers - doesn't bode well for the community. In any case, could this have been coming from contaminated "mate-pair" libraries (from large circularized fragments that are then fragmented themselves - fragments that contain the circ. point lead to outward facing read pairs that map R/F at long distances, while fragments that don't contain the circ. point are F/R pairs that map at short distances. I.e. all "mate-pair" libraries are contaminated to a certain extent with shorter-range "paired-end" reads. Now, given the opposite orientations, normally one or the other set won't map, but I think MAQ was able to align both, IIRC. HTH. ~J

05-18-2012, 02:59 AM	#4
rnaseek Member Location: USA Join Date: Nov 2011 Posts: 22	Does Illumina library's fragment length of 676 include adapter sequences? How big were the adaptor sequences?