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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...
Autores principales: | , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791352/ https://www.ncbi.nlm.nih.gov/pubmed/31616586 http://dx.doi.org/10.7717/peerj.7755 |
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author | Glenn, Travis C. Nilsen, Roger A. Kieran, Troy J. Sanders, Jon G. Bayona-Vásquez, Natalia J. Finger, John W. Pierson, Todd W. Bentley, Kerin E. Hoffberg, Sandra L. Louha, Swarnali Garcia-De Leon, Francisco J. del Rio Portilla, Miguel Angel Reed, Kurt D. Anderson, Jennifer L. Meece, Jennifer K. Aggrey, Samuel E. Rekaya, Romdhane Alabady, Magdy Belanger, Myriam Winker, Kevin Faircloth, Brant C. |
author_facet | Glenn, Travis C. Nilsen, Roger A. Kieran, Troy J. Sanders, Jon G. Bayona-Vásquez, Natalia J. Finger, John W. Pierson, Todd W. Bentley, Kerin E. Hoffberg, Sandra L. Louha, Swarnali Garcia-De Leon, Francisco J. del Rio Portilla, Miguel Angel Reed, Kurt D. Anderson, Jennifer L. Meece, Jennifer K. Aggrey, Samuel E. Rekaya, Romdhane Alabady, Magdy Belanger, Myriam Winker, Kevin Faircloth, Brant C. |
author_sort | Glenn, Travis C. |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-6791352 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-67913522019-10-15 Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) Glenn, Travis C. Nilsen, Roger A. Kieran, Troy J. Sanders, Jon G. Bayona-Vásquez, Natalia J. Finger, John W. Pierson, Todd W. Bentley, Kerin E. Hoffberg, Sandra L. Louha, Swarnali Garcia-De Leon, Francisco J. del Rio Portilla, Miguel Angel Reed, Kurt D. Anderson, Jennifer L. Meece, Jennifer K. Aggrey, Samuel E. Rekaya, Romdhane Alabady, Magdy Belanger, Myriam Winker, Kevin Faircloth, Brant C. PeerJ Bioinformatics 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. PeerJ Inc. 2019-10-11 /pmc/articles/PMC6791352/ /pubmed/31616586 http://dx.doi.org/10.7717/peerj.7755 Text en ©2019 Glenn et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Bioinformatics Glenn, Travis C. Nilsen, Roger A. Kieran, Troy J. Sanders, Jon G. Bayona-Vásquez, Natalia J. Finger, John W. Pierson, Todd W. Bentley, Kerin E. Hoffberg, Sandra L. Louha, Swarnali Garcia-De Leon, Francisco J. del Rio Portilla, Miguel Angel Reed, Kurt D. Anderson, Jennifer L. Meece, Jennifer K. Aggrey, Samuel E. Rekaya, Romdhane Alabady, Magdy Belanger, Myriam Winker, Kevin Faircloth, Brant C. Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext) |
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 | Bioinformatics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791352/ https://www.ncbi.nlm.nih.gov/pubmed/31616586 http://dx.doi.org/10.7717/peerj.7755 |
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