Hi everyone,
we have recently received Illumina reads from an RNA sample that has undergone oligodt capture and RT with template switching, followed by DSN normalization. These samples have a high 3' bias, when mapping the reads to flcDNAs generated by clonalization and sanger sequencing (i.e. full transcripts).
I was wondering if DSN normalization could be done avoiding the cDNA synthesis by template switching, that relies on oligodt capture. Has someone tried out an alternative protocol, like rRNA depletion, random primer cDNA synthesis and DSN normalization? Is this possible? I guess that something like this could improve the bias distribution of reads.
thank you
Carlos
we have recently received Illumina reads from an RNA sample that has undergone oligodt capture and RT with template switching, followed by DSN normalization. These samples have a high 3' bias, when mapping the reads to flcDNAs generated by clonalization and sanger sequencing (i.e. full transcripts).
I was wondering if DSN normalization could be done avoiding the cDNA synthesis by template switching, that relies on oligodt capture. Has someone tried out an alternative protocol, like rRNA depletion, random primer cDNA synthesis and DSN normalization? Is this possible? I guess that something like this could improve the bias distribution of reads.
thank you
Carlos