SAV % base question

[sophie mcquade]

I am new to looking at SAV. Would someone please explain to me why my % base would look like this. Thanks.

[Brian Bushnell]

Can you give more information about the library? I'm not sure, but it looks like a bar code, CCGGTTCA.

[sophie mcquade]

The library has a standard Illumina adapter on it. I am not sure why there is the change at around 60 bases either.

[Brian Bushnell]

Have you measured the insert size? They could be short. That wouldn't explain everything, but it might explain the change at around 60.

[sophie mcquade]

Here is what the library looks like. It was made using a protocol based on Khil et al Genome Res. 2012. 22: 957-965.

The abstract says: Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method—single-stranded DNA (ssDNA) sequencing (SSDS)—that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

[kmcarr]

I am new to looking at SAV. Would someone please explain to me why my % base would look like this. Thanks.

What you have is a library with a significant fraction of adapter dimer reads. If you closely examine the pattern of the predominant base call over the fist 60 bases of reads 1 and 2 you will find that they exactly match the sequence of the Illumina adapters. The adapters are each ~60nt long so when the read runs off the end of the adapter the sequencer has tendency to just call A's.

Now look at you Bioanalyzer trace. Do you see that teeny, tiny little peak at 125nt? That is your adapter dimer. Adapter dimer contaminations which appear insignificant on a BioA trace can account for a significant number of reads because they will cluster with much higher efficiency than your legitimate library molecules.

[Vinz]

And I guess you did put index information into your samplesheet. However, there was only one sample/one index sequenced. This should explain the pattern after 100 bases.

[sophie mcquade]

Thanks for all of your suggestions. I had asked the person analyzing the data if he was seeing adapter dimer reads and he said no. Now that I have the % base data I will have to ask him to recheck the data.

[HESmith]

Based on the relative peak heights, only 4-5% of your reads are adapter dimers. The effect on the data analysis would be minimal (a reduction in the fraction of aligned reads), which could easily have been missed by your informaticist.