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A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries

Yuguang Xiong, Magali Soumillon, Jie Wu, Jens Hansen, Bin Hu, Johan G.C. van Hasselt, Gomathi Jayaraman, Ryan Lim, Mehdi Bouhaddou, Loren Ornelas, Jim Bochicchio, Lindsay Lenaeus, Jennifer Stocksdale, Jaehee Shim, Emilda Gomez, Dhruv Sareen, Clive Svendsen, Leslie M. Thompson, Milind Mahajan, Ravi Iyengar, Eric A. Sobie, Evren U. Azeloglu, Marc R. Birtwistle
doi: https://doi.org/10.1101/098905
Yuguang Xiong
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Magali Soumillon
2Broad Institute of MIT and Harvard, Cambridge, MA
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Jie Wu
3Department of Biological Chemistry, University of California, Irvine, CA USA
4UCI MIND, University of California, Irvine, CA USA
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Jens Hansen
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Bin Hu
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Johan G.C. van Hasselt
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Gomathi Jayaraman
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Ryan Lim
3Department of Biological Chemistry, University of California, Irvine, CA USA
4UCI MIND, University of California, Irvine, CA USA
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Mehdi Bouhaddou
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Loren Ornelas
5Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
6iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Los Angeles, CA, USA
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Jim Bochicchio
2Broad Institute of MIT and Harvard, Cambridge, MA
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Lindsay Lenaeus
5Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
6iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Los Angeles, CA, USA
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Jennifer Stocksdale
4UCI MIND, University of California, Irvine, CA USA
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Jaehee Shim
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Emilda Gomez
5Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
6iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Los Angeles, CA, USA
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Dhruv Sareen
5Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
6iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Los Angeles, CA, USA
7Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
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Clive Svendsen
5Board of Governors-Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
6iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Los Angeles, CA, USA
7Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
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Leslie M. Thompson
3Department of Biological Chemistry, University of California, Irvine, CA USA
4UCI MIND, University of California, Irvine, CA USA
8Department of Psychiatry and Human Behavior, Neurobiology and Behavior, University of California, Irvine, CA USA
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Milind Mahajan
9Department of Genetics, Icahn School of Medicine at Mount Sinai, New York, NY 10029
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Ravi Iyengar
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Eric A. Sobie
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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Evren U. Azeloglu
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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  • For correspondence: evren.azeloglu@mssm.edu marc.birtwistle@mssm.edu
Marc R. Birtwistle
1Department of Pharmacological Sciences and DToxS LINCS Center, Icahn School of Medicine at Mount Sinai, New York, NY
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  • For correspondence: evren.azeloglu@mssm.edu marc.birtwistle@mssm.edu
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Abstract

Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 3’ end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 3’-digital gene expression (3’-DGE) approach with “conventional” random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 3’-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 06, 2017.
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A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries
Yuguang Xiong, Magali Soumillon, Jie Wu, Jens Hansen, Bin Hu, Johan G.C. van Hasselt, Gomathi Jayaraman, Ryan Lim, Mehdi Bouhaddou, Loren Ornelas, Jim Bochicchio, Lindsay Lenaeus, Jennifer Stocksdale, Jaehee Shim, Emilda Gomez, Dhruv Sareen, Clive Svendsen, Leslie M. Thompson, Milind Mahajan, Ravi Iyengar, Eric A. Sobie, Evren U. Azeloglu, Marc R. Birtwistle
bioRxiv 098905; doi: https://doi.org/10.1101/098905
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A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries
Yuguang Xiong, Magali Soumillon, Jie Wu, Jens Hansen, Bin Hu, Johan G.C. van Hasselt, Gomathi Jayaraman, Ryan Lim, Mehdi Bouhaddou, Loren Ornelas, Jim Bochicchio, Lindsay Lenaeus, Jennifer Stocksdale, Jaehee Shim, Emilda Gomez, Dhruv Sareen, Clive Svendsen, Leslie M. Thompson, Milind Mahajan, Ravi Iyengar, Eric A. Sobie, Evren U. Azeloglu, Marc R. Birtwistle
bioRxiv 098905; doi: https://doi.org/10.1101/098905

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