Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear (Ursus maritimus).

Frontiers in Genetics
Ashley ByrneChristopher Vollmers

Abstract

Transcriptome studies evaluating whole blood and tissues are often confounded by overrepresentation of highly abundant transcripts. These abundant transcripts are problematic, as they compete with and prevent the detection of rare RNA transcripts, obscuring their biological importance. This issue is more pronounced when using long-read sequencing technologies for isoform-level transcriptome analysis, as they have relatively lower throughput compared to short-read sequencers. As a result, long-read based transcriptome analysis is prohibitively expensive for non-model organisms. While there are off-the-shelf kits available for select model organisms capable of depleting highly abundant transcripts for alpha (HBA) and beta (HBB) hemoglobin, they are unsuitable for non-model organisms. To address this, we have adapted the recent CRISPR/Cas9-based depletion method (depletion of abundant sequences by hybridization) for long-read full-length cDNA sequencing approaches that we call Long-DASH. Using a recombinant Cas9 protein with appropriate guide RNAs, full-length hemoglobin transcripts can be depleted in vitro prior to performing any short- and long-read sequencing library preparations. Using this method, we sequenced depleted full-l...Continue Reading

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Citations

Jan 6, 2021·Nature Communications·Kristoffer Sahlin, Paul Medvedev
May 15, 2021·BMC Genomics·Shreya M BanerjeeLisa M Komoroske
May 18, 2021·The Journal of Biological Chemistry·Apple Cortez VollmersChristopher Vollmers

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Datasets Mentioned

BETA
GM12878
PRJNA514749

Methods Mentioned

BETA
RNA-seq
biopsies
pull-down
Illumina sequencing
Smart-seq2
in vitro transcription
Assay
PCR
Fluorescence

Software Mentioned

BLAST
kmer
Clustal Omega
Smart
BaseSpace
ImageJ
GENCODE
SamToPsl utility
C3POa
seq2

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