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Old 10-09-2015, 01:37 PM   #1
Alvaro Hernandez
Location: Champaign

Join Date: May 2009
Posts: 12
Default TruSeq Stranded mRNA HT kits, column 3 adaptors do not work with v4 chemistry?


We are experiencing an issue with the TruSeq® Stranded mRNA HT kit. We prepared three 96-well plates of RNAseq libraries in our EpMotion robots. The libraries are very consistent and have approximately the same concentration. We first sequenced one pool of 96 libraries on 8 lanes with single-reads 100nt, v4 chemistry. We saw that the libraries coming from column 3, which all have adaptor D703, had low number of reads and the reads from these libraries had low quality scores, whereas all the other libraries had great consistency in number of reads and quality.

The same thing happened with the other pools, always the libraries coming from this column did not perform well. Libraries were done in different robots, different months and different kit lot numbers.

We called Illumina and they asked us to sequence the same pools with the V3 chemistry and they provided the kits. And voila! All libraries sequenced beautifully.

There has been no resolution on this yet as we are only the 3rd lab to report this problem.

Has anyone else seen this issue?


Last edited by GenoMax; 10-10-2015 at 03:51 AM. Reason: Moving this post to a new thread. Contains new information that may be useful for others.
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Old 10-14-2015, 05:47 AM   #2
Martin Mikkelsen
Location: Denmark

Join Date: Jun 2010
Posts: 11

We have seen the same, but never really got an answer from Illumina
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Old 11-11-2015, 01:44 PM   #3
james hadfield
Cambridge, UK
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Location: Cambridge, UK

Join Date: Feb 2008
Posts: 221

Hi Alvaro and Martin,
We have seen drop out of the column 3 (D703) index on multiple RNA-seq runs. We are now very confident that this is not a simple problem. If we run the same library on multiple V4 HiSeq lanes we see the same problem, if we run the same library on MiSeq it is not present. If we run the same library on HiSeq rapid run it is not present. We are just testing on HiSeq 4000. I believe this is a HiSeq V4 issue and it appears that we can "fix" it by analysing the run without the chastity filter applied. This leads me to suspect the filter is throwing away more D703 indexes than the rest, possibly due to a contaminated adapter (maybe misincorportion during synthesis of that single oligo?

Our FAS has escalated this and Ilumina are testing. Have you had any notification back from your FAS on a fix for this?

1) Are you able to share details of your runs, the chemistry used and instrument?
2) Are you able to go back and repeat analysis without the chastity filter?
3) Have you looked to see if there is any biological affect of the lower read number?

I'd really appreciate getting a conversation going off SEQanswers on this to go into it in some depth. WOuld you both be able to get in touch directly?
You can contact me at
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