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
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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