RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery
Abstract
Accurate genome annotations are essential in modern biology and biotechnology, yet they are still largely based on genome sequencing and comparative analyses. We show that the Clostridium acetobutylicum genome annotation can be markedly improved by integrating bioinformatic predictions with RNA sequencing (RNAseq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth conditions. Analysis of an initial assembly revealed errors due to background signals and limitations of assembly algorithms. Hurdles for RNAseq transcriptome mapping include optimizing library complexity and sequencing depth, yet most studies report low sequencing depth and ignore the effect of ribosomal RNA abundance. An integrative analysis was developed to combine motif predictions, single‐nucleotide resolution sequencing depth, and library complexity to resolve difficulties in assembly curation. This minimized false positive error and determined gene boundaries, in some cases, to the exact base‐pair of prior studies. This is the first strand‐specific transcriptome assembly in a Clostridium organism. © 2018 American Institute of Chemical Engineers AIChE J , 64: 4271–4280, 2018
- Publication Date:
- Research Org.:
- Univ. of Delaware, Newark, DE (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC)
- OSTI Identifier:
- 1610933
- Alternate Identifier(s):
- OSTI ID: 1475227
- Grant/Contract Number:
- SC0007092; DE‐SC0007092
- Resource Type:
- Accepted Manuscript
- Journal Name:
- AIChE Journal
- Additional Journal Information:
- Journal Volume: 64; Journal Issue: 12; Journal ID: ISSN 0001-1541
- Publisher:
- American Institute of Chemical Engineers
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Engineering; RNAseq; novel transcripts; acetone-butanol; small RNAs; antisense RNAs
Citation Formats
Ralston, Matthew T., and Papoutsakis, Eleftherios T. RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery. United States: N. p., 2022.
Web. doi:10.1002/aic.16396.
Ralston, Matthew T., & Papoutsakis, Eleftherios T. RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery. United States. https://doi.org/10.1002/aic.16396
Ralston, Matthew T., and Papoutsakis, Eleftherios T. Tue .
"RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery". United States. https://doi.org/10.1002/aic.16396. https://www.osti.gov/servlets/purl/1610933.
@article{osti_1610933,
title = {RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery},
author = {Ralston, Matthew T. and Papoutsakis, Eleftherios T.},
abstractNote = {Accurate genome annotations are essential in modern biology and biotechnology, yet they are still largely based on genome sequencing and comparative analyses. We show that the Clostridium acetobutylicum genome annotation can be markedly improved by integrating bioinformatic predictions with RNA sequencing (RNAseq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth conditions. Analysis of an initial assembly revealed errors due to background signals and limitations of assembly algorithms. Hurdles for RNAseq transcriptome mapping include optimizing library complexity and sequencing depth, yet most studies report low sequencing depth and ignore the effect of ribosomal RNA abundance. An integrative analysis was developed to combine motif predictions, single‐nucleotide resolution sequencing depth, and library complexity to resolve difficulties in assembly curation. This minimized false positive error and determined gene boundaries, in some cases, to the exact base‐pair of prior studies. This is the first strand‐specific transcriptome assembly in a Clostridium organism. © 2018 American Institute of Chemical Engineers AIChE J , 64: 4271–4280, 2018},
doi = {10.1002/aic.16396},
journal = {AIChE Journal},
number = 12,
volume = 64,
place = {United States},
year = {Tue Aug 30 00:00:00 EDT 2022},
month = {Tue Aug 30 00:00:00 EDT 2022}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 5 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Comprehensive Transcriptome Analysis of the Periodontopathogenic Bacterium Porphyromonas gingivalis W83
journal, October 2011
- Hovik, H.; Yu, W. -H.; Olsen, I.
- Journal of Bacteriology, Vol. 194, Issue 1
Sequence and arrangement of genes encoding enzymes of the acetone-production pathway of Clostridium acetobutylicum ATCC 824
journal, January 1993
- Petersen, Daniel J.; Cary, Jeffrey W.; Vanderleyden, Jos
- Gene, Vol. 123, Issue 1
Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile
journal, May 2013
- Soutourina, Olga A.; Monot, Marc; Boudry, Pierre
- PLoS Genetics, Vol. 9, Issue 5
Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum
journal, May 2018
- Jones, Alexander J.; Fast, Alan G.; Clupper, Michael
- Applied and Environmental Microbiology, Vol. 84, Issue 14
Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene
journal, November 2001
- Harris, Lm; Blank, L.; Desai, Rp
- Journal of Industrial Microbiology and Biotechnology, Vol. 27, Issue 5
Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.
journal, February 1994
- Nair, R. V.; Bennett, G. N.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 176, Issue 3
Northern, Morphological, and Fermentation Analysis of spo0A Inactivation and Overexpression in Clostridium acetobutylicum ATCC 824
journal, July 2002
- Harris, L. M.; Welker, N. E.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 184, Issue 13
Assessing De Novo transcriptome assembly metrics for consistency and utility
journal, January 2013
- O’Neil, Shawn T.; Emrich, Scott J.
- BMC Genomics, Vol. 14, Issue 1
Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis
journal, March 2012
- Nicolas, P.; Mader, U.; Dervyn, E.
- Science, Vol. 335, Issue 6072
Bias detection and correction in RNA-Sequencing data
journal, January 2011
- Zheng, Wei; Chung, Lisa M.; Zhao, Hongyu
- BMC Bioinformatics, Vol. 12, Issue 1
The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation
journal, January 2015
- Al-Hinai, Mohab A.; Jones, Shawn W.; Papoutsakis, Eleftherios T.
- Microbiology and Molecular Biology Reviews, Vol. 79, Issue 1
Validation of two ribosomal RNA removal methods for microbial metatranscriptomics
journal, September 2010
- He, Shaomei; Wurtzel, Omri; Singh, Kanwar
- Nature Methods, Vol. 7, Issue 10
An efficient rRNA removal method for RNA sequencing in GC-rich bacteria
journal, January 2013
- Peano, Clelia; Pietrelli, Alessandro; Consolandi, Clarissa
- Microbial Informatics and Experimentation, Vol. 3, Issue 1
Trimmomatic: a flexible trimmer for Illumina sequence data
journal, April 2014
- Bolger, Anthony M.; Lohse, Marc; Usadel, Bjoern
- Bioinformatics, Vol. 30, Issue 15
Transcriptional organization of the Clostridium acetobutylicum genome
journal, March 2004
- Paredes, C. J.
- Nucleic Acids Research, Vol. 32, Issue 6
Induction of heat shock proteins during initiation of solvent formation inClostridium acetobutylicum
journal, September 1990
- Pich, Andreas; Narberhaus, Franz; Bahl, Hubert
- Applied Microbiology and Biotechnology, Vol. 33, Issue 6
RNA-seq: An assessment of technical reproducibility and comparison with gene expression arrays
journal, July 2008
- Marioni, J. C.; Mason, C. E.; Mane, S. M.
- Genome Research, Vol. 18, Issue 9
Stress- and Growth Phase-Associated Proteins of Clostridium acetobutylicum
journal, August 1988
- Terracciano, Joseph S.; Rapaport, Eliezer; Kashket, Eva R.
- Applied and Environmental Microbiology, Vol. 54, Issue 8
Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
journal, January 2012
- Giannoukos, Georgia; Ciulla, Dawn M.; Huang, Katherine
- Genome Biology, Vol. 13, Issue 3
Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis.
journal, November 1993
- Fischer, R. J.; Helms, J.; Dürre, P.
- Journal of Bacteriology, Vol. 175, Issue 21
Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum
journal, August 2001
- Nolling, J.; Breton, G.; Omelchenko, M. V.
- Journal of Bacteriology, Vol. 183, Issue 16
The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain.
journal, September 1997
- Cornillot, E.; Nair, R. V.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 179, Issue 17
Small RNAs in the Genus Clostridium
journal, December 2010
- Chen, Y.; Indurthi, D. C.; Jones, S. W.
- mBio, Vol. 2, Issue 1
Full-length transcriptome assembly from RNA-Seq data without a reference genome
journal, May 2011
- Grabherr, Manfred G.; Haas, Brian J.; Yassour, Moran
- Nature Biotechnology, Vol. 29, Issue 7
The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum
journal, October 1995
- Bennett, George N.; Rudolph, Frederick B.
- FEMS Microbiology Reviews, Vol. 17, Issue 3
Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
journal, August 2012
- Nejepinska, Jana; Malik, Radek; Moravec, Martin
- PLoS ONE, Vol. 7, Issue 8
D³ Data-Driven Documents
journal, December 2011
- Bostock, M.; Ogievetsky, V.; Heer, J.
- IEEE Transactions on Visualization and Computer Graphics, Vol. 17, Issue 12
Sequencing depth and coverage: key considerations in genomic analyses
journal, January 2014
- Sims, David; Sudbery, Ian; Ilott, Nicholas E.
- Nature Reviews Genetics, Vol. 15, Issue 2
TransRate: reference-free quality assessment of de novo transcriptome assemblies
journal, June 2016
- Smith-Unna, Richard; Boursnell, Chris; Patro, Rob
- Genome Research, Vol. 26, Issue 8
Metabolite stress and tolerance in the production of biofuels and chemicals: Gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum
journal, January 2010
- Alsaker, Keith V.; Paredes, Carlos; Papoutsakis, Eleftherios T.
- Biotechnology and Bioengineering
Structure and Complexity of a Bacterial Transcriptome
journal, March 2009
- Passalacqua, K. D.; Varadarajan, A.; Ondov, B. D.
- Journal of Bacteriology, Vol. 191, Issue 10
The transcriptional program underlying the physiology of clostridial sporulation
journal, January 2008
- Jones, Shawn W.; Paredes, Carlos J.; Tracy, Bryan
- Genome Biology, Vol. 9, Issue 7
Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production.
journal, September 1994
- Nair, R. V.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 176, Issue 18
mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis.
journal, January 1992
- Gerischer, U.; Dürre, P.
- Journal of Bacteriology, Vol. 174, Issue 2
Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program
journal, August 2003
- Tomas, C. A.; Welker, N. E.; Papoutsakis, E. T.
- Applied and Environmental Microbiology, Vol. 69, Issue 8
Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq
journal, September 2011
- Wang, Yi; Li, Xiangzhen; Mao, Yuejian
- BMC Genomics, Vol. 12, Issue 1
The primary transcriptome of the major human pathogen Helicobacter pylori
journal, February 2010
- Sharma, Cynthia M.; Hoffmann, Steve; Darfeuille, Fabien
- Nature, Vol. 464, Issue 7286
Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria
journal, August 2012
- Raghavan, Rahul; Sloan, Daniel B.; Ochman, Howard
- mBio, Vol. 3, Issue 4
A comparative genomic view of clostridial sporulation and physiology
journal, October 2005
- Paredes, Carlos J.; Alsaker, Keith V.; Papoutsakis, Eleftherios T.
- Nature Reviews Microbiology, Vol. 3, Issue 12
Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling
journal, January 2013
- Li, Shan; Dong, Xia; Su, Zhengchang
- BMC Genomics, Vol. 14, Issue 1
Differential expression of a Clostridium acetobutylicum antisense RNA: implications for regulation of glutamine synthetase.
journal, December 1992
- Fierro-Monti, I. P.; Reid, S. J.; Woods, D. R.
- Journal of Bacteriology, Vol. 174, Issue 23
Comprehensive comparative analysis of strand-specific RNA sequencing methods
journal, August 2010
- Levin, Joshua Z.; Yassour, Moran; Adiconis, Xian
- Nature Methods, Vol. 7, Issue 9
Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.
journal, January 1989
- Wiesenborn, D. P.; Rudolph, F. B.; Papoutsakis, E. T.
- Applied and Environmental Microbiology, Vol. 55, Issue 2
Widespread Antisense Transcription in Escherichia coli
journal, May 2010
- Dornenburg, James E.; DeVita, Anne M.; Palumbo, Michael J.
- mBio, Vol. 1, Issue 1
Biases in Illumina transcriptome sequencing caused by random hexamer priming
journal, April 2010
- Hansen, Kasper D.; Brenner, Steven E.; Dudoit, Sandrine
- Nucleic Acids Research, Vol. 38, Issue 12
Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing
journal, January 2012
- Robles, José A.; Qureshi, Sumaira E.; Stephen, Stuart J.
- BMC Genomics, Vol. 13, Issue 1
RNA-Seq: a revolutionary tool for transcriptomics
journal, January 2009
- Wang, Zhong; Gerstein, Mark; Snyder, Michael
- Nature Reviews Genetics, Vol. 10, Issue 1
Accurate and comprehensive sequencing of personal genomes
journal, July 2011
- Ajay, S. S.; Parker, S. C. J.; Ozel Abaan, H.
- Genome Research, Vol. 21, Issue 9
Preliminary Modeling of Transfer RNA Kinetics in the Cytoplasm of <I>Escherichia coli</I> Bacteria
journal, March 2010
- Piros, Sándor J.; Husseini, Ghaleb A.
- Advanced Science Letters, Vol. 3, Issue 1
Predicting the molecular complexity of sequencing libraries
journal, February 2013
- Daley, Timothy; Smith, Andrew D.
- Nature Methods, Vol. 10, Issue 4
Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.
journal, May 1992
- Narberhaus, F.; Giebeler, K.; Bahl, H.
- Journal of Bacteriology, Vol. 174, Issue 10
Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum
journal, June 2015
- Venkataramanan, Keerthi P.; Min, Lie; Hou, Shuyu
- Biotechnology for Biofuels, Vol. 8, Issue 1
Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum
journal, March 2004
- Tomas, C. A.; Beamish, J.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 186, Issue 7
Accurate whole human genome sequencing using reversible terminator chemistry
journal, November 2008
- Bentley, David R.; Balasubramanian, Shankar; Swerdlow, Harold P.
- Nature, Vol. 456, Issue 7218
Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism
journal, July 2012
- Cai, Xianpeng; Servinsky, Matthew; Kiel, James
- Applied Microbiology and Biotechnology, Vol. 97, Issue 10
Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress
journal, January 2013
- Wang, Qinghua; Venkataramanan, Keerthi; Huang, Hongzhan
- BMC Systems Biology, Vol. 7, Issue 1
The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum
journal, January 2013
- Venkataramanan, Keerthi P.; Jones, Shawn W.; McCormick, Kevin P.
- BMC Genomics, Vol. 14, Issue 1
Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum.
journal, December 1990
- Gerischer, U.; Dürre, P.
- Journal of Bacteriology, Vol. 172, Issue 12
Transcriptional Program of Early Sporulation and Stationary-Phase Events in Clostridium acetobutylicum
journal, September 2005
- Alsaker, K. V.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 187, Issue 20
Next-generation transcriptome assembly
journal, September 2011
- Martin, Jeffrey A.; Wang, Zhong
- Nature Reviews Genetics, Vol. 12, Issue 10
Transcriptional Analysis of spo0A Overexpression in Clostridium acetobutylicum and Its Effect on the Cell's Response to Butanol Stress
journal, March 2004
- Alsaker, K. V.; Spitzer, T. R.; Papoutsakis, E. T.
- Journal of Bacteriology, Vol. 186, Issue 7
An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803
journal, January 2011
- Mitschke, Jan; Georg, Jens; Scholz, Ingeborg
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 5
Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor
journal, January 1999
- Nair, Ramesh V.; Green, Edward M.; Watson, David E.
- Journal of Bacteriology, Vol. 181, Issue 1
Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.
journal, May 1992
- Narberhaus, F.; Bahl, H.
- Journal of Bacteriology, Vol. 174, Issue 10
The Sequence Alignment/Map format and SAMtools
journal, June 2009
- Li, H.; Handsaker, B.; Wysoker, A.
- Bioinformatics, Vol. 25, Issue 16
ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia
journal, September 2012
- Landt, S. G.; Marinov, G. K.; Kundaje, A.
- Genome Research, Vol. 22, Issue 9
Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824.
journal, June 1996
- Boynton, Z. L.; Bennet, G. N.; Rudolph, F. B.
- Journal of Bacteriology, Vol. 178, Issue 11
Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum
text, January 2009
- Gerischer, Ulrike; Dürre, Peter
- Universität Ulm
TransRate: reference-free quality assessment of de novo transcriptome assemblies.
text, January 2016
- Smith-Unna, Richard; Boursnell, Chris; Patro, Rob
- Apollo - University of Cambridge Repository