Hello,
does anyone have experience with a much lower number of reads than expected from RNAseq using the NextSeq Illumina system? I prepared cDNA libraries from RNA isolated from glioma cell cultures (Zymogen RNA isolation kit), then I did rRNA depletion (Ribocop kit), polyA tailing, RNase R treatment (all to purify circRNAs) and then I prepared cDNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina. I used UMI while preparing libraries. Based on quantification of cDNA libraries, the expected number of reads was 20-25 million but it ended up at 5-7 million reads. Do you have any suggestions why this could happen? Is it possible that the library did not cluster and do you have any idea why? After that, I also used KAPA kit for Illumina to check if the adapters have been ligated and according to this kit they were. For RNAseq NextSeq 500/550 High Output Kit v2.5 (75 cycles) was used.
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Have you checked the sequencing run metrics? Nothing suspect? I'd suggest you contact Illumina support, they are always very helpful.
David Aciole Barbosa Thank you, I have checked it with the employees of the genomic core facility and a bioinformatician, nothing suspect was there. But I'm gonna contact Illumina support, thank you for the idea!
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