Adapterama II: Universal Amplicon Sequencing on Illumina Platforms (TaggiMatrix)

Details

• Personal Author:
  Bayona-Vásquez NJ ; Bentley KE ; Bian X ; Cummings BS ; Faircloth BC ; Finger JW ; Gao B ; Gilbert GS ; Glenn TC ; Guo TL ; Hoffberg SL ; Hubbell SP ; Kieran TJ ; Kolli RT ; Lash LH ; Lefever DE ; Louha S ; Lu K ; Mauricio R ; McKee AM ; Molina M ; Pierson TW ; Rothrock MJ Jr. ; Rushmore J ; Shaw TI ; Thomas JC IV ; Wong K
• Description:
  Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 50 end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 x 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadrupleindexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. [Description provided by NIOSH]
• Subjects:
  Genetics Microbiology Occupational Health Public Health Safety
• Keywords:
  Author Keywords: MiSeq Bioinformatics Biological Factors Deoxyribonucleic Acids DNA Fusion Primers Genomics Hierarchical Indexing Internal Tagging Libraries Life Sciences Multiplexing Next Generation Sequencing PCR Public Health Quadruple Indexing Sampling

  More +
• ISSN:
  2167-8359
• Document Type:
  Text
• Genre:
  Journal Article
• Place as Subject:
  Alabama ; California ; Georgia ; Louisiana ; Michigan ; New York ; North Carolina ; OSHA Region 2 ; OSHA Region 3 ; OSHA Region 4 ; OSHA Region 5 ; OSHA Region 6 ; OSHA Region 9 ; Pennsylvania ; Tennessee
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  7
• NIOSHTIC Number:
  nn:20057702
• Citation:
  PeerJ 2019 Oct; 7:e7786
• Contact Point Address:
  Travis C. Glenn, Department of Environmental Health Science, University of Georgia, Athens, GA
• Email:
  travisg@uga.edu
• Federal Fiscal Year:
  2020
• Peer Reviewed:
  True
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:e2ba3209a87b5d8d8146b1f1364fef3bfb40368ab35c75a2bd49eb5f3bb394b4272f904112ecefcd7a100034d04b3b272ce191158f66f8ac9b07e60cb1c9d676
• Download URL:
  https://stacks.cdc.gov/view/cdc/214954/cdc_214954_DS1.pdf
• File Type:
  [PDF - 388.88 KB ]