The 24th Annual Meeting of the RNA Society will take place from June 11th to June 16th in Krakow, Poland. Lexogen is delighted to welcome you there as a Gold Sponsor, continuing its long-standing support of the RNA Society’s annual meetings and RNA Salons.
Find the Lexogen booth at the exhibition area, attend our poster, join us for a breakfast seminar, and get a chance to win prizes!
Applying Lexogen RNA-Seq technologies to determine transcription & mRNA turnover, RNA-protein interactions, and poly(A)+ & poly(A)– RNA 3’ end abundances
Thursday, June 13, 2019
07:45 – 08:45 am
Park Inn by Radisson Krakow
The breakfast will be served at 07:30 am
Chairperson: Lukas Paul (Senior Manager of Scientific Affairs, Lexogen, Vienna, Austria)
While NGS-based RNA sequencing has established itself as a powerful tool for transcriptome analysis, it entails diverse platforms, applications, and protocols. In this seminar, three stories will be presented that reveal how selected technologies from Lexogen – SLAMseq metabolic RNA labeling, the CORALL total RNA-Seq library preparation protocol, and the QuantSeq 3’ RNA-Seq method – can be applied to distinct research questions.
The seminar will take place in Park Inn by Radisson Krakow, a one-minute walk from the ICE Krakow Congress Centre.
Presented during the Wednesday poster session at 20:30 – 23:00.
Cap-dependent linker ligation increases specificity for full-length products compared to template switch reaction
Lexogen GmbH, Vienna, Austria
Pamela Moll1, Musashi Tsujita1, Florian Kabinger1, Tomas Dozd1, Michael Ante1, Andreas Tuerk1, Torsten Reda1, and Alexander Seitz1
1 Lexogen GmbH, Campus Vienna Biocenter 5, 1030 Vienna, Austria
Nanopore enables full-length sequencing of RNA or cDNA. A fast and easy way to obtain full-length cDNA is the commonly used template switch reaction. The reverse transcriptase (RT) adds non-templated nucleotides (preferentially C’s) at the end of a transcript which hybridize to abundant template switch oligos. However, non-templated nucleotides can also be added to fragmented RNAs or premature termination sites of the RT. Artificial Spike-in transcripts are an essential part to monitoring the quality of an RNA-Seq experiment, to control NGS sample preparation, base callers and algorithms by adding a ground truth to the NGS experiment. We used Lexogen SIRV™ set 3 containing 69 Spike-In RNA Variant controls, simulating alternative splicing of 7 SIRV genes plus antisense transcription, plus 92 ERCC Spike in controls (External RNA Controls Consortium Spike-In controls, Thermo Fisher Scientific Inc.). ERCCs are monoexonic but cover a concentration range of 6 orders of magnitude. TeloPrime is a full-length cDNA preparation kit offered by Lexogen. Exceptional 5‘-Cap specificity is achieved with the proprietary CAP dependent linker ligation. We capped SIRV set 3 using the Vaccinia Capping Enzyme and protocol from NEB (M2080). Universal Human Reference RNA (UHRR) was spiked in with the capped SIRV set 3 before the sample was subject to a controlled degradation. Nanopore sequencing libraries were made either by using the template switching protocol or a modified TeloPrime protocol (v3) from intact and degraded RNA aliquots. The new TeloPrime v3 Nanopore libraries contain a 12 nt unique molecular Index (UMI) that is introduced with the RT primer, enabling to account for sequencer and PCR errors in high coverage NGS data. Degraded RNA resulted in shorter libraries for the less Cap-sensitive template switching protocol, but little delay in the PCR cycles, while for TeloPrime v3 degraded RNA libraries resulted in significantly less amplifiable library. The analyses of apparent transcript start site distributions by Nanopore sequencing showed a higher cap specificity for TeloPrime v3 than for the template switching protocol. Hence, TeloPrime v3 enables an increased accuracy for transcript 5’ end detection.
Keywords: Cap Specificity, Full-length cDNA, nanopore sequencing
Win a prize!
Resolve the Lexogen puzzle and get a chance to win one of 5 air loungers.
You can find the puzzle and the sticker in the attendee’s bag or get them at the Lexogen booth.
Looking forward to meeting you soon.
Your Lexogen RNA 2019 team!