We are seeing very low quality for the first few bases of the 2nd end in paired-end sequencing on the HiSeq. We have data from 6-7 experiments where the library prep and sequencing was conducted at 3 different sequencing centers around the country. The library preps varied between centers, but each center did the sequencing on HiSeq 2000s. It seems like there is a problem with chemistry on the sequencer.
We were told by one sequencing center:
"It turned out that there was a NaOH problem…the protocol uses NaOH to strip of the index prior to sequencing the 2nd read. While the NaOH reagent sat on the machine, for some reason, it degraded and proper removal of the index was not achieved. Adding fresh NaOH a day before index2 did the trick. The Qscore looks amazing...."
Another sequencing center said:
"They [Illumina] have suggested that NaOH is not working well anymore to denature index1 away. If this is not complete, it will continue sequencing 7 dark cycles after index1 and then 8nt from the adapter, which is exactly the adapter sequence that is the top match in my 8mer analysis."
I'm attaching an image of what our poor quality scores look like for the 2nd end; the first end looks great. I'd like to hear if anyone else is seeing the same problem and whether you have a similar or different answer from Illumina or your sequencing centers.
Apologies if you're seeing this post a second time. One Senior Member of SeqAnswers suggested that I post this as a new thread. I had previously attached it to a thread about a similar problem on the miSeq, which could very well be due to a different problem.
We were told by one sequencing center:
"It turned out that there was a NaOH problem…the protocol uses NaOH to strip of the index prior to sequencing the 2nd read. While the NaOH reagent sat on the machine, for some reason, it degraded and proper removal of the index was not achieved. Adding fresh NaOH a day before index2 did the trick. The Qscore looks amazing...."
Another sequencing center said:
"They [Illumina] have suggested that NaOH is not working well anymore to denature index1 away. If this is not complete, it will continue sequencing 7 dark cycles after index1 and then 8nt from the adapter, which is exactly the adapter sequence that is the top match in my 8mer analysis."
I'm attaching an image of what our poor quality scores look like for the 2nd end; the first end looks great. I'd like to hear if anyone else is seeing the same problem and whether you have a similar or different answer from Illumina or your sequencing centers.
Apologies if you're seeing this post a second time. One Senior Member of SeqAnswers suggested that I post this as a new thread. I had previously attached it to a thread about a similar problem on the miSeq, which could very well be due to a different problem.
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