I was wondering how many basepairs are usually sequenced with mRNA/cDNA illumina methods from upstream of the polyedenlation site? Thanks.

  • $\begingroup$ No one can answer this. There are too many sequencing methods. $\endgroup$
    – swbarnes2
    Jul 23, 2018 at 20:44

1 Answer 1


50-75 bp could be achieved.

Refrencing TruSeq® RNA Sample Preparation v2 Guide. My lab used random primers and read lengths were an average of 75 bp in length for an RNA seq project. But controlling distance from polyA wasn't considered in out project.

Using oglio(dT) primers you could try and control a specific length from the polyA tail.Two basic variations use either random primers or oligo(dT) primers for this reaction. Oligo(dT) primers are highly 3’ biased and mostly suitable for mRNA abundance (expression) analysis. Random primers also result in some bias, which can be reduced by fragmentation of the input RNA.

Source: RNA Sequencing Methods Collection - Illumina, Page 8.

Different lengths can also be achieved for different needs. Gene expression / RNA Profiling – Quantifying the coding transcriptome typically requires a short single read (often 50–75 bp) to minimize reading across splice junctions while counting all RNAs in the pool.

Transcriptome Analysis – Novel transcriptome assembly and annotation projects tend to benefit from longer, paired-end reads (such as 2 x 75 bp) to enable more complete coverage of the transcripts and identification of novel variants or splice sites. Paired-end reads are required to get information from both 5’ and 3’ ends of RNA species with Stranded RNA-Seq library preparation kits.

Small RNA Analysis – Due to the short length of small RNA, a single read (usually a 50 bp read) usually covers the entire sequence. A read length of 50 bp sequences most small RNAs, plus enough of the adapter to be accurately identified and trimmed during data analysis.

Source: Illumina (Considerations for RNA-Seq read length and coverage)


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