Improving eukaryotic genome annotation using single molecule mRNA sequencing
Abstract Background The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum...
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doaj-f4dff008f2f0405383530c7aad84b2672020-11-24T21:16:08ZengBMCBMC Genomics1471-21642018-03-0119111410.1186/s12864-018-4555-7Improving eukaryotic genome annotation using single molecule mRNA sequencingVincent Magrini0Xin Gao1Bruce A. Rosa2Sean McGrath3Xu Zhang4Kymberlie Hallsworth-Pepin5John Martin6John Hawdon7Richard K. Wilson8Makedonka Mitreva9McDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineDepartment of Microbiology, Immunology and Tropical Medicine, The George Washington UniversityMcDonnell Genome Institute, Washington University School of MedicineMcDonnell Genome Institute, Washington University School of MedicineAbstract Background The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq. Results We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features. Conclusion Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies.http://link.springer.com/article/10.1186/s12864-018-4555-7Genome annotation improvementPacific bioscience mRNA sequencingAncylostoma ceylanicumHookwormGene loci |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Vincent Magrini Xin Gao Bruce A. Rosa Sean McGrath Xu Zhang Kymberlie Hallsworth-Pepin John Martin John Hawdon Richard K. Wilson Makedonka Mitreva |
spellingShingle |
Vincent Magrini Xin Gao Bruce A. Rosa Sean McGrath Xu Zhang Kymberlie Hallsworth-Pepin John Martin John Hawdon Richard K. Wilson Makedonka Mitreva Improving eukaryotic genome annotation using single molecule mRNA sequencing BMC Genomics Genome annotation improvement Pacific bioscience mRNA sequencing Ancylostoma ceylanicum Hookworm Gene loci |
author_facet |
Vincent Magrini Xin Gao Bruce A. Rosa Sean McGrath Xu Zhang Kymberlie Hallsworth-Pepin John Martin John Hawdon Richard K. Wilson Makedonka Mitreva |
author_sort |
Vincent Magrini |
title |
Improving eukaryotic genome annotation using single molecule mRNA sequencing |
title_short |
Improving eukaryotic genome annotation using single molecule mRNA sequencing |
title_full |
Improving eukaryotic genome annotation using single molecule mRNA sequencing |
title_fullStr |
Improving eukaryotic genome annotation using single molecule mRNA sequencing |
title_full_unstemmed |
Improving eukaryotic genome annotation using single molecule mRNA sequencing |
title_sort |
improving eukaryotic genome annotation using single molecule mrna sequencing |
publisher |
BMC |
series |
BMC Genomics |
issn |
1471-2164 |
publishDate |
2018-03-01 |
description |
Abstract Background The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq. Results We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features. Conclusion Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies. |
topic |
Genome annotation improvement Pacific bioscience mRNA sequencing Ancylostoma ceylanicum Hookworm Gene loci |
url |
http://link.springer.com/article/10.1186/s12864-018-4555-7 |
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