A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes
High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target g...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2015-07-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00731/full |
| _version_ | 1811239767420960768 |
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| author | Craig W. Herbold Claus ePelikan Orest eKuzyk Bela eHausmann Roey eAngel David eBerry Alexander eLoy |
| author_facet | Craig W. Herbold Claus ePelikan Orest eKuzyk Bela eHausmann Roey eAngel David eBerry Alexander eLoy |
| author_sort | Craig W. Herbold |
| collection | DOAJ |
| description | High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse and high quality sets of amplicon sequence data for modern studies in microbial ecology. |
| first_indexed | 2024-04-12T13:06:54Z |
| format | Article |
| id | doaj.art-4bd461706c944bebab62aedfbba96354 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-12T13:06:54Z |
| publishDate | 2015-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-4bd461706c944bebab62aedfbba963542022-12-22T03:32:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2015-07-01610.3389/fmicb.2015.00731150975A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genesCraig W. Herbold0Claus ePelikan1Orest eKuzyk2Bela eHausmann3Roey eAngel4David eBerry5Alexander eLoy6University of ViennaUniversity of ViennaUniversity of ViennaUniversity of ViennaUniversity of ViennaUniversity of ViennaUniversity of ViennaHigh throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse and high quality sets of amplicon sequence data for modern studies in microbial ecology.http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00731/full16S rRNAamoAdsrAnifHfunctional genedsrB |
| spellingShingle | Craig W. Herbold Claus ePelikan Orest eKuzyk Bela eHausmann Roey eAngel David eBerry Alexander eLoy A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes Frontiers in Microbiology 16S rRNA amoA dsrA nifH functional gene dsrB |
| title | A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| title_full | A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| title_fullStr | A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| title_full_unstemmed | A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| title_short | A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| title_sort | flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes |
| topic | 16S rRNA amoA dsrA nifH functional gene dsrB |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00731/full |
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