Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets
Introduction Whole-metagenome sequencing can be a rich source of information about the structure and function of entire metagenomic communities, but getting accurate and reliable results from these datasets can be challenging. Analysis of these datasets is founded on the mapping of sequencing reads...
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| Format: | Article |
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PeerJ Inc.
2019-01-01
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| Series: | PeerJ |
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| Online Access: | https://peerj.com/articles/6174.pdf |
| _version_ | 1797420531708854272 |
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| author | Paul Greenfield Nai Tran-Dinh David Midgley |
| author_facet | Paul Greenfield Nai Tran-Dinh David Midgley |
| author_sort | Paul Greenfield |
| collection | DOAJ |
| description | Introduction Whole-metagenome sequencing can be a rich source of information about the structure and function of entire metagenomic communities, but getting accurate and reliable results from these datasets can be challenging. Analysis of these datasets is founded on the mapping of sequencing reads onto known genomic regions from known organisms, but short reads will often map equally well to multiple regions, and to multiple reference organisms. Assembling metagenomic datasets prior to mapping can generate much longer and more precisely mappable sequences but the presence of closely related organisms and highly conserved regions makes metagenomic assembly challenging, and some regions of particular interest can assemble poorly. One solution to these problems is to use specialised tools, such as Kelpie, that can accurately extract and assemble full-length sequences for defined genomic regions from whole-metagenome datasets. Methods Kelpie is a kMer-based tool that generates full-length amplicon-like sequences from whole-metagenome datasets. It takes a pair of primer sequences and a set of metagenomic reads, and uses a combination of kMer filtering, error correction and assembly techniques to construct sets of full-length inter-primer sequences. Results The effectiveness of Kelpie is demonstrated here through the extraction and assembly of full-length ribosomal marker gene regions, as this allows comparisons with conventional amplicon sequencing and published metagenomic benchmarks. The results show that the Kelpie-generated sequences and community profiles closely match those produced by amplicon sequencing, down to low abundance levels, and running Kelpie on the synthetic CAMI metagenomic benchmarking datasets shows similar high levels of both precision and recall. Conclusions Kelpie can be thought of as being somewhat like an in-silico PCR tool, taking a primer pair and producing the resulting ‘amplicons’ from a whole-metagenome dataset. Marker regions from the 16S rRNA gene were used here as an example because this allowed the overall accuracy of Kelpie to be evaluated through comparisons with other datasets, approaches and benchmarks. Kelpie is not limited to this application though, and can be used to extract and assemble any genomic region present in a whole metagenome dataset, as long as it is bound by a pairs of highly conserved primer sequences. |
| first_indexed | 2024-03-09T07:03:48Z |
| format | Article |
| id | doaj.art-0af9d58582c34a75929d888b7b6cf0d4 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T07:03:48Z |
| publishDate | 2019-01-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-0af9d58582c34a75929d888b7b6cf0d42023-12-03T09:45:59ZengPeerJ Inc.PeerJ2167-83592019-01-016e617410.7717/peerj.6174Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasetsPaul Greenfield0Nai Tran-Dinh1David Midgley2Commonwealth Scientific and Industrial Research Organisation, North Ryde, NSW, AustraliaCommonwealth Scientific and Industrial Research Organisation, North Ryde, NSW, AustraliaCommonwealth Scientific and Industrial Research Organisation, North Ryde, NSW, AustraliaIntroduction Whole-metagenome sequencing can be a rich source of information about the structure and function of entire metagenomic communities, but getting accurate and reliable results from these datasets can be challenging. Analysis of these datasets is founded on the mapping of sequencing reads onto known genomic regions from known organisms, but short reads will often map equally well to multiple regions, and to multiple reference organisms. Assembling metagenomic datasets prior to mapping can generate much longer and more precisely mappable sequences but the presence of closely related organisms and highly conserved regions makes metagenomic assembly challenging, and some regions of particular interest can assemble poorly. One solution to these problems is to use specialised tools, such as Kelpie, that can accurately extract and assemble full-length sequences for defined genomic regions from whole-metagenome datasets. Methods Kelpie is a kMer-based tool that generates full-length amplicon-like sequences from whole-metagenome datasets. It takes a pair of primer sequences and a set of metagenomic reads, and uses a combination of kMer filtering, error correction and assembly techniques to construct sets of full-length inter-primer sequences. Results The effectiveness of Kelpie is demonstrated here through the extraction and assembly of full-length ribosomal marker gene regions, as this allows comparisons with conventional amplicon sequencing and published metagenomic benchmarks. The results show that the Kelpie-generated sequences and community profiles closely match those produced by amplicon sequencing, down to low abundance levels, and running Kelpie on the synthetic CAMI metagenomic benchmarking datasets shows similar high levels of both precision and recall. Conclusions Kelpie can be thought of as being somewhat like an in-silico PCR tool, taking a primer pair and producing the resulting ‘amplicons’ from a whole-metagenome dataset. Marker regions from the 16S rRNA gene were used here as an example because this allowed the overall accuracy of Kelpie to be evaluated through comparisons with other datasets, approaches and benchmarks. Kelpie is not limited to this application though, and can be used to extract and assemble any genomic region present in a whole metagenome dataset, as long as it is bound by a pairs of highly conserved primer sequences.https://peerj.com/articles/6174.pdfMetagenomesAmpliconsTargeted assemblyCommunity structureIn-silico PCR |
| spellingShingle | Paul Greenfield Nai Tran-Dinh David Midgley Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets PeerJ Metagenomes Amplicons Targeted assembly Community structure In-silico PCR |
| title | Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets |
| title_full | Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets |
| title_fullStr | Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets |
| title_full_unstemmed | Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets |
| title_short | Kelpie: generating full-length ‘amplicons’ from whole-metagenome datasets |
| title_sort | kelpie generating full length amplicons from whole metagenome datasets |
| topic | Metagenomes Amplicons Targeted assembly Community structure In-silico PCR |
| url | https://peerj.com/articles/6174.pdf |
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