HiSeq4000 small RNA libraries (vs 2500)

[tjhu11]

Hi All,

I'm having some issues running a custom small RNA sequencing protocol on the HiSeq4000, when the same library works perfectly fine on the 2500.

Illumina may not have tested the TruSeq Small RNA protocol using the 4000, so I was wondering if anyone in the community has successfully done sequenced small RNAs on the 4000.

Thanks!

[jwfoley]

I can't help you, but can you tell us what issues you had and whether you'd already tried the same libraries on a different machine?

[tjhu11]

Should've been more specific, yes the libraries are simply not clustering! and they do cluster on the 2500. But I'm just wondering if it is due to the minor differences (2-3nt shorter) in the clustering arms or whether the size of the whole molecule is important in the 4000 due to the differences in clustering.

[sirisha]

Hope you have solved your problem already. If not....small RNA will not cluster on 4000 as the fragment size need to be atleast 300bp to cluster on 4000.

[luc]

Even adapter dimers (insert size 0) cluster and sequence just fine on the Hiseq 4000 - there is no problem with the insert length. Small RNA libraries can work. The OP problems might relate to the "custom" part.

[kmcarr]

Originally Posted by tjhu11

Hi All,

I'm having some issues running a custom small RNA sequencing protocol on the HiSeq4000, when the same library works perfectly fine on the 2500.

Illumina may not have tested the TruSeq Small RNA protocol using the 4000, so I was wondering if anyone in the community has successfully done sequenced small RNAs on the 4000.

Thanks!

The HiSeq4000 product pages do not list TruSeq Small RNA libraries as one of the supported library types for the instrument. Likewise the TruSeq Small RNA support documents do not list the HiSeq4000 as a supported platform.

TruSeq Small RNA libraries require different sequencing and indexing primers than other TruSeq libraries (e.g. DNA, mRNA, etc.). The HiSeq2500 clustering and sequencing reagent kits (both Rapid and High Output) include all required primers for all TruSeq library types as well as Nextera libraries.

While Illumina has not chosen to share with me personally I would bet my favorite P20 Pipetman* that the TruSeq Small RNA primers are not included in the HiSeq4000 reagent kits. If true then your observation that the libraries worked on the 2500 but not the 4000 is entirely expected. Always check kit & platform compatibility before loading anything on a sequencer.

*Since I rarely if ever do actual bench work anymore its not much of bet really.

[luc]

If the Illumina kit indeed does not work on the HS 4000 - this one does: http://www.biooscientific.com/Next-G...rary-Prep-Kits

[kmcarr]

Originally Posted by luc

If the Illumina kit indeed does not work on the HS 4000 - this one does: http://www.biooscientific.com/Next-G...rary-Prep-Kits

Has this been tested and confirmed? Without addition of custom sequencing and index primers?

The Bioo NEXTflex Small RNA Kits produce libraries with the same adapter sequences as the TruSeq Small RNA Kit. If you have successfully sequenced Bioo NEXTflex small RNA libraries on a HiSeq 4000, using the stock clustering/sequencing reagent kits, then I'll gladly retract my earlier supposition that the 4000 kits lack the Small RNA primers.

[kerplunk412]

I contacted a collaborator today who has been using Bioo Scientific small RNA kits and just had samples sequenced on a HiSeq 4000, and although they haven't analyzed the data in detail yet everything appears normal on first pass. I will update if they run into any problems. As kmcarr mentioned, the adapter sequences in the Bioo kits are identical to TruSeq (except Bioo's contain random bases to reduce ligase bias), so since the Bioo kit worked then TruSeq should as well.

As a disclaimer, I work for Bioo Scientific.

[helskel]

Custom primers don't work on the HiSeq 4000. Were you trying to use custom primers?

[LSAD]

Our TruSeq Small RNA library works very well on HiSeq 4000. It is not listed as compatible with HiSeq4000 on Illumina website because the protocol is prone to produce adapter dimer. Check bioanalyzer and make sure there is no even trace of adapter dimer.