Number of "good" reads

[strob]

Hello all,

When you do a solexa run, how many reads can you obtain using for example 1 lane (let's say for reads of 35 bp or 50 bp)? And more important, how many of these reads are useful, meaning that they don't contain any adapter/primer sequence?

Does it also differ depending on the type of analysis you want to perform (RNASeq, WGS, Gene enrichment, ...) or do you always see the same amount of reads containing adapter sequences?

I ask this because in certain datasets, we see that almost 2/3 of the reads contain an adapter sequence, which is huge.

Thanks for the comments,

Steven

[strob]

no one any experience?

[nilshomer]

We do not see this. In fact, I would say it is definitely less than 1%.

[elaney_k]

I have seen similarly high % of adapter sequences in samples where the the adapter-input ratio at the ligation stage of sample prep was not titrated properly, especially low input libraries like ChIP-seq. For most libraries, if you run some of your prepped library on a 2% gel/bioanalyser you should be able to spot adapter-dimer contamination (bright band at ~110bp).
To rectify the adapter contamination problem I had (with a ChIP-seq sample) I diluted the Illumina adapter 1:50 instead of 1:10 as stated in the manual and the subsequent sequencing run produced data with <1% adapter sequences.

What type of datasets are you seeing with large amounts of adapter sequences?

[strob]

For the moment, we saw it when we did a gene enrichment sequencing analysis (selected genomic DNA).

Thanks for your comment

[elaney_k]

gene enrichment sequencing analysis (selected genomic DNA).

What was your gene enrichment method? Did you enrich for your target after sequencing library prep? If so you would not expect to see any adapter sequences in your reads.
If you did your gene enrichment method before library prep, it would be possible to see reads with adapter sequences for the same reason I saw them with my samples, i.e. with low input amount + poor titration of adapters in ligation step.

[strob]

enrichment before library prep, so we have the same problems

We will test your solution

many thanks

[elaney_k]

I'd recommend doing the library first then enriching as oligos from on-array enrichment methods have been know to leak from the array into your sample and then you have the problem of sample contamination (assuming you're enriching using arrays). At least if you do the library prep first then after the enrichment step carry out a second (or multiple parallel) enrichment PCRs you will only be enriching for samples that have the adapters ligated and leaked oligos won't be an issue.