Adapterama I: Universal Stubs and Primers for Thousands of Dual-Indexed Illumina Libraries (iTru & iNext)

@article{Glenn2016AdapteramaIU,
  title={Adapterama I: Universal Stubs and Primers for Thousands of Dual-Indexed Illumina Libraries (iTru \& iNext)},
  author={Travis C. Glenn and Roger A. Nilsen and Troy J. Kieran and John W. Finger and Todd W. Pierson and Kerin Bentley and Sandra L. Hoffberg and Swarnali Louha and Francisco Javier Garc{\'i}a-De Le{\'o}n and Miguel Angel del Rio Portilla and Kurt D. Reed and Jennifer L. Anderson and Jennifer K Meece and Samuel E. Aggery and Romdhane Rekaya and Magdy S. Alabady and Myriam B{\'e}langer and Kevin Winker and Brant C. Faircloth},
  journal={bioRxiv},
  year={2016}
}
Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing the time and costs associated with: 1) start-up (i.e., doing NGS for the first time), 2) buy-in (i.e., getting any data from a run), and 3) sample preparation. Although many researchers have focused on reducing sample preparation costs, few have addressed the first two problems. Here, we present iTru and iNext, dual-indexing systems for Illumina libraries that help address all three of… 
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111 Citations
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111 Citations

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This work presents a low-cost and highly robust approach for the construction of dual-digest RADseq libraries and presents eight adapter designs that work with 72 restriction enzyme combinations, and demonstrates the approach by the use of one set of adapters and oneSet of restriction enzymes across a variety of non-model organisms to discover thousands of variable loci in each species.

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Chapter 3 Targeted DNA Region Re-sequencing

All methods are usually paired with DNA sequence tags (also known as barcodes, indexes, or molecular identifi ers, MID tags; see Faircloth and Glenn 2012 ) to identify individual samples from a pool of samples.

RADcap: sequence capture of dual‐digest RADseq libraries with identifiable duplicates and reduced missing data

RADcap is introduced, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches.

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Practical considerations for plant phylogenomics

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Gathering genetic data for rare species is one of the biggest remaining obstacles in modern phylogenetics, particularly for megadiverse groups such as arthropods. Next generation sequencing…

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52 References

Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about $1USD per Sample (3RAD)

This work presents a low-cost and highly robust approach for the construction of dual-digest RADseq libraries and presents eight adapter designs that work with 72 restriction enzyme combinations, and demonstrates the approach by the use of one set of adapters and oneSet of restriction enzymes across a variety of non-model organisms to discover thousands of variable loci in each species.

Adapterama IV: Sequence Capture of Dual-digest RADseq Libraries with Identifiable Duplicates (RADcap)

RADcap is demonstrated, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches.

Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

This work has developed a method that reveals the rate of sample misidentification within current multiplex sequencing experiments and identified the occurrence of mixed clusters on the flow as the predominant source of error.

Field guide to next‐generation DNA sequencers

  • T. Glenn
  • Biology
    Molecular ecology resources
  • 2011
T careful thought about the desired characteristics of these systems is warranted before purchasing or using any of them, and the major characteristics of each commercially available platform are summarized.

Illumina sequencing library preparation for highly multiplexed target capture and sequencing.

This protocol describes a fast and reliable method for the preparation of barcoded ("indexed") sequencing libraries for Illumina's Genome Analyzer platform, which avoids expensive library preparation kits and can be performed in a 96-well plate setup using multi-channel pipettes, requiring not more than two or three days of lab work.

RADcap: sequence capture of dual‐digest RADseq libraries with identifiable duplicates and reduced missing data

RADcap is introduced, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches.

Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of GC-biased genomes

An amplification-free method of library preparation is presented, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly.

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The critical role of sequencing library quality is examined and important challenges when preparing NGS libraries from DNA and RNA sources are considered, including the quantity and physical characteristics of the RNA or DNA source material.

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This work presents an approach in which 192 sequencing libraries can be produced in a single day of technician time at a cost of about $15 per sample, effective not only for low-pass whole-genome sequencing, but also for simultaneously enriching them in pools of approximately 100 individually barcoded samples for a subset of the genome.

Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

It is concluded that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.
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