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Can I please ask for advice on how best to compare, on paper first of all, setting up an RNAseq experiment and the relative pros and cons of Illumina short read sequencing versus Oxford nanopore long read sequencing.
From the literature there appears far fewer reads for the latter but these reads are much longer and provide information on splice variants.
Previously we have done RNAseq by Illumina kits and we performed a rRNA depletion on human total RNA (5 ug) then a TruSeq stranded total RNA kit prep then onto a NovaSeq6000 with 4 biological replicates and acquired ~40 M reads for each. What would this depth possibly look like in an ONT experimental setup is my question?
Using ONT approaches and attempting isoform detection in mRNA transcripts then either PCR-cDNA Sequencing Kit (SQK-PCS111) or Direct cDNA Sequencing Kit (SQK-DCS109) seems to be the way to go for RNAseq. The choice seems to comes down to how much starting RNA you have and if you want to avoid PCR of the cDNA and what your output needs are. PCR-cDNA sequencing (SQK-PCS111) gives the highest output of reads and requires 4 ng of PolyA mRNA and has a newer chemistry which reduces intron priming during reverse transcription. Direct cDNA sequencing (SQK-DCS109) avoids PCR amplification of cDNA but requires 100 ng PolyA+ RNA and doesn't have the newer generation chemistry of SQK-PCS111.
Many thanks in advance for any advice or comments or kindly pointing me in the right direction in the literature!
From the literature there appears far fewer reads for the latter but these reads are much longer and provide information on splice variants.
Previously we have done RNAseq by Illumina kits and we performed a rRNA depletion on human total RNA (5 ug) then a TruSeq stranded total RNA kit prep then onto a NovaSeq6000 with 4 biological replicates and acquired ~40 M reads for each. What would this depth possibly look like in an ONT experimental setup is my question?
Using ONT approaches and attempting isoform detection in mRNA transcripts then either PCR-cDNA Sequencing Kit (SQK-PCS111) or Direct cDNA Sequencing Kit (SQK-DCS109) seems to be the way to go for RNAseq. The choice seems to comes down to how much starting RNA you have and if you want to avoid PCR of the cDNA and what your output needs are. PCR-cDNA sequencing (SQK-PCS111) gives the highest output of reads and requires 4 ng of PolyA mRNA and has a newer chemistry which reduces intron priming during reverse transcription. Direct cDNA sequencing (SQK-DCS109) avoids PCR amplification of cDNA but requires 100 ng PolyA+ RNA and doesn't have the newer generation chemistry of SQK-PCS111.
Many thanks in advance for any advice or comments or kindly pointing me in the right direction in the literature!