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buthercup_ch 07-26-2016 01:12 AM

HiSeq 4000 upgrade

We have been running RNA-Seq in HiSeq2000/2500 and are quite happy with the results.
But there is now just available the HiSeq4000.

Please, any one having experience with the new sequencer? Is that such a great improvement of the data? compared with the additional cost?

Thanks for any advice

GenoMax 07-26-2016 03:37 AM

Improvement is going to be in the amount of data you get. Q-scores are not a big issue with good libraries now-a-days.

Consider this important point: your libraries would need to meet more stringent criteria for insert size (350-450 bp) to avoid optical duplicates (due to pad hopping). If you have shorter or longer inserts then you may be better off staying with 2000/2500.

GW_OK 07-26-2016 07:00 AM

Also if you do any bisulfite work or anything with low base diversity the 4000 is not nearly as robust as the 2500.

buthercup_ch 07-26-2016 10:29 PM

Thanks GenoMax for your comments.

The actual size of the insert is 300 bp, what we have been using with 2000/2500.
Please can you explain in more details why the insert size is constraint in such a way?
And what are "pad-hopping" and "optical duplicates"?

Thanks in advance

PS. For the record, we are working with microbial transcriptomes.

GenoMax 07-27-2016 03:49 AM

See this blog post by James Hadfield for answers to your questions and some addition information.

"Pad hopping" refers to contamination of nearby nanowells which may lead to mixed clusters or at worst identical sequence clusters in physically close nanowells.

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