Directional sequencing and strandedness for TruSeq Stranded Total RNA and mRNA libraries
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Why is stranded information useful? Directional sequence (stranded) information is crucial in the identification of antisense transcripts for overlapping genes, and may also increase the percentage of uniquely alignable reads in poorly-annotated species. Having directional information also eases the alignment and assembly processes for bioinformatics analyses.
Is there any reason to not have stranded information? Projects that are in progress may choose to keep their current configuration and workflow, rather than changing their protocols in the middle of the study.
How do the TruSeq Stranded mRNA and Total RNA protocols generate stranded cDNA? Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second-strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. Further specificity is achieved by the addition of Actinomycin D to the First Strand Master Mix Act D (FSA). Actinomycin prevents spurious DNA-dependent synthesis during first-strand synthesis while allowing RNA-dependent synthesis.
How is strandedness maintained after DNA amplification? Strandedness is maintained via the directionality of the adapters. The p7 adapter will be on the 3' end of the cDNA strand. As a consequence, the cDNA strand is sequenced.
Which strand is sequenced in TruSeq Stranded mRNA and Total RNA libraries? Read 1 maps to the antisense strand, and read 2 maps to the sense strand. Note: this is not applicable for the TruSeq Small RNA Kit, which is also stranded, but has a different workflow. With TruSeq Small RNA libraries, Read 1 maps to the sense strand.
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