Abstract Detail

Recent Topics Posters

Peredo , Elena Lopez [1], Cardon, Zoe [2], Thomas, Suzanne [3], Bruce, Douglas [4].

Library construction using RNA customized target removal for simultaneous nuclear and organelle expression profiling in species of the green microalgae Scenedesmus.

One of the challenges of RNA-seq methods is to ensure reliable and sufficient sequencing depth of biologically relevant transcripts. RNA extraction and library prep methods have an important impact in sequencing results and therefore in the final interpretation of the data. Our investigations on the responses to slow desiccation and fast rehydration of the green microalgae Scenedesmus (Chlorophyta) required of simultaneous analysis of nuclear and organelle gene expression profiles but also, removal of ribosomic RNA (rRNA) of diverse origin. In our RNA-seq experiments, we generated paired-end strand-specific cDNA libraries and used sequence-based, customized, enzymatic targeted removal of rRNA (InDA-C, Nugen) during library preparation. We avoided methods enriching for mRNA that take advantage of features of eukaryotic mature coding transcripts, such as poly(A) tails to prevent biasing against recovery of chloroplast and mitochondrial transcripts. Due to the prokaryotic nature of chloroplast and mitochondria, organelle transcripts lack some of the features characteristic of nuclear mature transcripts such as poly(A) tails. If present, these poly(A) tails are usually associated with signaling for mRNA degradation. However, rRNA can constitute up to 90% of total RNA extracted swamping any sequencing effort so rRNA removal strategies is essential. In the case of green algae cultures, where native bacteria are usually required for sustained growth, rRNA removal is especially problematic because the simultaneous presence of rRNA of eukaryotic (nuclear) and prokaryotic (chloroplast, mitochondria and bacteria) origin. Using a combination of commercially available primers targeting plant and prokaryotic rRNA and custom made primers specific to nuclear and chloroplast rRNA regions of Scenedesmus rotundus we were able to simultaneously remove algal and bacterial rRNA from our samples. These custom made primers designed for one species successfully removed rRNA in other two closely related species of S. costatus and S. deserticola. After optimization, nuclear rRNA has been estimated in ~10% of our reads, while the presence of prokaryotic rRNA is ~3%. Nearly 50% of the total reads aligned to contigs containing coding regions of plant origin (identified by blastx to nr NCBI database).

1 - Marine Biological Laboratories, MBL St 7, Starr Building, Woods Hole, MA, 02543-1015, USA
2 - Marine Biological Laboratory, The Ecosystems Center, 7 MBL St, Woods Hole, MA, 02543-1015, USA
3 - Marine Biological Laboratory, The Ecosystems Center, 7 MBL St, Woods Hole, MA, 02543-1015, USA
4 - Brock University, Department of Biological Sciences, 500 Glenridge Ave, St. Catharines, ON, ON L2S 3A1, Canada

Keywords:
library prep
Ribosomic removal
RNA-seq.

Presentation Type: Recent Topics Poster
Session: P, Recent Topics Posters
Location: Exhibit Hall/Savannah International Trade and Convention Center
Date: Monday, August 1st, 2016
Time: 5:30 PM
Number: PRT031
Abstract ID:1224
Candidate for Awards:None