Illumina Hiseq 3000 / enough coverage for RNAseq analysis

[sheibani]

Dear all,

I want to sequence total RNA samples from Human on Hiseq 3000 for the first time. Do you have any idea how many samples per lane will give me enough coverage to perform transcriptomics and splicing analysis.
I was used to do it with 0.1 sample per lane with Hiseq 2500.

Many thanks in advance for your help.
Regards,
Sheibani

[SylvainL]

I was used to do it with 0.1 sample per lane with Hiseq 2500.

Hi Sheibani,

you mean you were using 10 lanes for 1 sample? Around 2 billions reads per sample????

[sheibani]

Hi,
No, I mean 0.1 lane per sample (10 sample per lane) in HiSeq 2500.

Thanks,

[SylvainL]

Ok, I started to think your pipeline was really not improved

[sheibani]

So, Do you have any idea about number of samples per lane for Hiseq 3000?

Thanks

[GenoMax]

There is roughly 300 million clusters per lane (passing filter) that one can expect to get from a well performing patterned flowcell. That output is achieved with a couple of caveats. Your libraries need to conform to a much stricter QC profile (inserts < 500 bp and very small %, preferably close to 0% of adapter dimers/small fragments). You can then multiply that number with cycles/type of sequencing you are going to use to decide the number of samples you can multiplex.

[SylvainL]

Sorry, I never analyzed data from HiSeq3000, but according to Illumina datasheet, it seems you can expect a bit more than with the 2500. I think if I were you, I would first try with the same number of samples per lane...

[nucacidhunter]

It seems that you were doing analysis with 20-25M reads/sample. With HiSeq 3000 output you can do 12-13 samples. You need to check with sequencing service provider number of guranteed reads/lane to figure out the exact number.

[sheibani]

Many many thanks for your help and prompt reply. I got some idea about it, I am going to contact the sequencing service provider to get more information from them too.

Kind Regards,