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Just finished developing our method for targeted quantitative RNA sequencing. Our studies use Ion torrent platform, but this approach works well on Illumina as well. Our unpublished data shows our results are concordant between the two platforms as well. These latter studies will be out in 2014. These studies are part of a larger FDA initiative for concordance between RNA sequencing studies.
Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.
Regards, Tom Blomquist
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Hi guys, we have trying to use v2 kits for Bacterial transcriptimics. All values during library preparation seems good but final output is really low, with 50/60% of polyclonals. It seems that the step generating problems is the onetouch.
Anyone have any suggestion? any alternative protocols?
Thanks to anyone that will help us!
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Originally posted by Gorbenzer View PostHi guys, we have trying to use v2 kits for Bacterial transcriptimics. All values during library preparation seems good but final output is really low, with 50/60% of polyclonals. It seems that the step generating problems is the onetouch.
Anyone have any suggestion? any alternative protocols?
Thanks to anyone that will help us!
This can be frustrating.
Check out the methods section in our paper, and we describe our approach to avoiding the heterodimerization issue which leads to polyclonal reads.
Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.
-Tom Blomquist
Comment
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Originally posted by Gorbenzer View PostHi guys, we have trying to use v2 kits for Bacterial transcriptimics. All values during library preparation seems good but final output is really low, with 50/60% of polyclonals. It seems that the step generating problems is the onetouch.
Anyone have any suggestion? any alternative protocols?
Thanks to anyone that will help us!
Comment
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We have about 30 successful RNAseq runs on the Ion Torrent Proton using the RNAseq v2 kit with polyA selected RNA. One modification we have made is to size select a narrower range (110-200bp) using the Pippin system. We have found that this results in a higher "effective" library concentration when using the Bioanalyzer quantification range as suggested in the RNAseq kit manual (ie more of our final library is in the optimal size range for sequencing). We had to lower the concentration from the recommended 11pM to ~5pM to keep the polyclonal % under control and have been able to get up to 88M reads (average is more like 72M). The recommended concentration has been changed in the OT2 200 v3 manual but we have lowered our input accordingly and are getting similar results.
I would suggest lowering your concentration by ~0.5-1pM each run and monitoring from there.
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Hi guys,
We have about 10 successful run for RNA seq on the proton. We use the RNA-seq v2 kit from Life tech. Both Ribo- and Ribo gold and RNaseIII fragmentation and (chemical from NEB). We also have the AB lib builder...which was not so successful at first but getting better....but 13 libs in a batch is a little slow.
Our polyclonality is in average about 30%...
MBZMG1 what are you getting for polyclonality?...we are sequencing with the V3 kit.
NEXTGENSEQ can you provide more info about the NuGen library kits (links and what metrics are you using to define the NuGen kit as 'working better')?
Tx.
RemitoAmigo
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Originally posted by RemitoAmigo View PostMBZMG1 what are you getting for polyclonality?...we are sequencing with the V3 kit.
On another note -what are people using for alignment? We have found that Bowtie2/Tophat2 do not play well with Ion Torrent data and have had much better success with GSNAP.
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