Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
A NEW NGS LIBRARY PREPARATION METHOD FOR
TRANSCRIPTOME PROFILING WITH ENHANCED
SENSITIVITY OF TRANSCRIPT DETECTION
Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.16)
Daniela Munafo Erbay Yigit Deyra Rodriguez Mehmet Karaca Keerthana Krishnan Pingfang
Liu Lynne Apone Vaishnavi Panchapakesa Laurie Mazzola Joanna Bybee Danielle Rivizzigno
Fiona Stewart Eileen Dimalanta Theodore Davis
New England Biolabs, Inc.
RNA‐seq (RNA sequencing) is a transcriptome‐profiling method that uses next generation sequencing.
It is widely used for genome‐wide expression analysis as well as detection of mutations, fusion
transcripts, alternative splicing, and post‐transcriptional modifications. RNA‐seq is becoming increasingly
common in molecular diagnostics; providing better insights into how altered transcripts
impact the biological pathways and the molecular mechanisms associated with disease progression.
The successful adoption of RNA‐seq into the molecular diagnostics will depend on the library preparation
techniques that require low input RNA, and can capture the entire molecular repertoire within
a sample without sequence bias.
Here, we present a high efficiency method for strand‐specific RNA‐seq that retains information about
which strand of DNA is transcribed. Determining the polarity of RNA transcripts is important for the correct
annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for
correct determination of gene expression levels in the presence of antisense transcripts. This method is
based on the labeling and excision of the second strand cDNA, and it is compatible with both poly A‐
tail enriched and ribosome‐depleted RNA. Our results show this improved method generates significantly
higher library yields that enable use of lower amounts of input RNA. More‐ over, our new method results in
increased sensitivity and specificity, especially for low‐abundance transcripts, reduced PCR duplicates and
sequence bias, delivering high quality strand‐specific data. This streamlined protocol is also amenable to
large‐scale library construction and automation.
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