Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)
Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2019-10-01
|
| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/7755.pdf |
| _version_ | 1797424331391762432 |
|---|---|
| author | Travis C. Glenn Roger A. Nilsen Troy J. Kieran Jon G. Sanders Natalia J. Bayona-Vásquez John W. Finger Todd W. Pierson Kerin E. Bentley Sandra L. Hoffberg Swarnali Louha Francisco J. Garcia-De Leon Miguel Angel del Rio Portilla Kurt D. Reed Jennifer L. Anderson Jennifer K. Meece Samuel E. Aggrey Romdhane Rekaya Magdy Alabady Myriam Belanger Kevin Winker Brant C. Faircloth |
| author_facet | Travis C. Glenn Roger A. Nilsen Troy J. Kieran Jon G. Sanders Natalia J. Bayona-Vásquez John W. Finger Todd W. Pierson Kerin E. Bentley Sandra L. Hoffberg Swarnali Louha Francisco J. Garcia-De Leon Miguel Angel del Rio Portilla Kurt D. Reed Jennifer L. Anderson Jennifer K. Meece Samuel E. Aggrey Romdhane Rekaya Magdy Alabady Myriam Belanger Kevin Winker Brant C. Faircloth |
| author_sort | Travis C. Glenn |
| collection | DOAJ |
| description | Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms. |
| first_indexed | 2024-03-09T07:59:42Z |
| format | Article |
| id | doaj.art-820e6176c0ea47cda321efffde5448c4 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T07:59:42Z |
| publishDate | 2019-10-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-820e6176c0ea47cda321efffde5448c42023-12-03T00:47:58ZengPeerJ Inc.PeerJ2167-83592019-10-017e775510.7717/peerj.7755Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)Travis C. Glenn0Roger A. Nilsen1Troy J. Kieran2Jon G. Sanders3Natalia J. Bayona-Vásquez4John W. Finger5Todd W. Pierson6Kerin E. Bentley7Sandra L. Hoffberg8Swarnali Louha9Francisco J. Garcia-De Leon10Miguel Angel del Rio Portilla11Kurt D. Reed12Jennifer L. Anderson13Jennifer K. Meece14Samuel E. Aggrey15Romdhane Rekaya16Magdy Alabady17Myriam Belanger18Kevin Winker19Brant C. Faircloth20Department of Environmental Health Science, University of Georgia, Athens, GA, United States of AmericaGeorgia Genomics and Bioinformatics Core, University of Georgia, Athens, GA, United States of AmericaDepartment of Environmental Health Science, University of Georgia, Athens, GA, United States of AmericaDepartment of Pediatrics, School of Medicine, University of California San Diego, La Jolla, CA, United States of AmericaDepartment of Environmental Health Science, University of Georgia, Athens, GA, United States of AmericaDepartment of Environmental Health Science, University of Georgia, Athens, GA, United States of AmericaDepartment of Environmental Health Science, University of Georgia, Athens, GA, United States of AmericaDepartment of Genetics, University of Georgia, Athens, GA, United States of AmericaDepartment of Genetics, University of Georgia, Athens, GA, United States of AmericaInstitute of Bioinformatics, University of Georgia, Athens, GA, United States of AmericaLaboratorio de Genética para la Conservación, Centro de Investigaciones Biológicas del Noroeste, SC, Instituto Politécnico Nacional, La Paz, MexicoCentro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, MexicoDepartment of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, United States of AmericaIntegrated Research and Development Laboratory, Marshfield Clinic Research Institute, Marshfield, WI, United States of AmericaIntegrated Research and Development Laboratory, Marshfield Clinic Research Institute, Marshfield, WI, United States of AmericaInstitute of Bioinformatics, University of Georgia, Athens, GA, United States of AmericaInstitute of Bioinformatics, University of Georgia, Athens, GA, United States of AmericaGeorgia Genomics and Bioinformatics Core, University of Georgia, Athens, GA, United States of AmericaGeorgia Genomics and Bioinformatics Core, University of Georgia, Athens, GA, United States of AmericaUniversity of Alaska Museum, Fairbanks, AK, United States of AmericaDepartment of Biological Sciences and Museum of Natural Science, Louisiana State University, Baton Rouge, LA, United States of AmericaMassively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.https://peerj.com/articles/7755.pdfIlluminaNext Generation SequencingNovaSeqSample PreparationPoolingMultiplexing |
| spellingShingle | Travis C. Glenn Roger A. Nilsen Troy J. Kieran Jon G. Sanders Natalia J. Bayona-Vásquez John W. Finger Todd W. Pierson Kerin E. Bentley Sandra L. Hoffberg Swarnali Louha Francisco J. Garcia-De Leon Miguel Angel del Rio Portilla Kurt D. Reed Jennifer L. Anderson Jennifer K. Meece Samuel E. Aggrey Romdhane Rekaya Magdy Alabady Myriam Belanger Kevin Winker Brant C. Faircloth Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) PeerJ Illumina Next Generation Sequencing NovaSeq Sample Preparation Pooling Multiplexing |
| title | Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
| title_full | Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
| title_fullStr | Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
| title_full_unstemmed | Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
| title_short | Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
| title_sort | adapterama i universal stubs and primers for 384 unique dual indexed or 147 456 combinatorially indexed illumina libraries itru inext |
| topic | Illumina Next Generation Sequencing NovaSeq Sample Preparation Pooling Multiplexing |
| url | https://peerj.com/articles/7755.pdf |
| work_keys_str_mv | AT traviscglenn adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT rogeranilsen adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT troyjkieran adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT jongsanders adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT nataliajbayonavasquez adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT johnwfinger adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT toddwpierson adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT kerinebentley adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT sandralhoffberg adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT swarnalilouha adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT franciscojgarciadeleon adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT miguelangeldelrioportilla adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT kurtdreed adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT jenniferlanderson adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT jenniferkmeece adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT samueleaggrey adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT romdhanerekaya adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT magdyalabady adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT myriambelanger adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT kevinwinker adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext AT brantcfaircloth adapteramaiuniversalstubsandprimersfor384uniquedualindexedor147456combinatoriallyindexedilluminalibrariesitruinext |