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Old 09-02-2018, 01:29 AM   #1
fxjonas
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Default Low quality sequencing from too short R1

Hi,

Our lab started using the mcSCRBseq protocol for RNA library preparation (https://www.protocols.io/view/mcscrb...otocol-p9kdr4w), which recommends a 16/8/0/50 read settings in the Illumina machine 16= BC+ UMI, 50= cDNA. Interestingly, the read quality with these settings became significantly worse than when we used our previous libraries with 50/8/8/16. (% >Q30 dropped from 85-95% to 65-75% and increased phrasing 0.1 -> 0.3). After contacting Illumina about this problem, they mentioned that a too short R1 could cause problems when determining good clusters and result in problems later on. This sounds very conclusive, but unfortunately, we are not able to adjust the length of R1 and actually test his prediction. Therefore I was wondering, if anyone has experience with short Read1 sequencing and if someone knows a way to improve the quality of the reads.
Thanks for your help,
Felix
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Old 09-02-2018, 04:53 AM   #2
GenoMax
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You just have to live with the quality of the reads you get. Illumina requires 25 cycles in first read to accurately calibrate Q scores. Your type of sequencing is common with "Drop-seq" like methods where the first read is short as in your case.

I am not sure if the read structure in your case can allow you to sequence longer on read 1. If it can you could sequence 25 cycles and then drop the 7 bases.
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