Emulsion PCR-coupled target enrichment: An effective fishing method for high-throughput sequencing of poorly preserved ancient DNA

Kihana, Makio; Mizuno, Fuzuki; Sawafuji, Rikai; Wang, Li; Ueda, Shintaroh

doi:10.1016/j.gene.2013.07.040

Cited by 19 publications

(10 citation statements)

References 26 publications

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“…However, it is difficult to rule out inter-locus amplification bias as the origin of this pattern; only two samples (Mammoths 9 and 10) show low correlations between regional duplication rate and unique coverage depth, and for these samples unique enrichment rates do not correspond to bait properties. That amplification biases appear to dictate coverage patterns in this data set clearly encourages the use of amplification-minimal techniques (15, 42, 43) with less biased DNA polymerases (40, 44) or emulsion PCR (45). …”

Section: Resultsmentioning

confidence: 68%

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

2013

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Targeted DNA enrichment through hybridization capture (EHC) is rapidly replacing PCR as the method of choice for enrichment prior to genomic resequencing. This is especially true in the case of ancient DNA (aDNA) from long-dead organisms, where targets tend to be highly fragmented and outnumbered by contaminant DNA. However, the behavior of EHC using aDNA has been quite variable, making success difficult to predict and preventing efficient sample equilibration during multiplexed sequencing runs. Here, we evaluate whether quantitative PCR (qPCR) measurements of aDNA samples correlate with on-target read counts before and after EHC. Our data indicate that not only do simple target qPCRs correlate strongly with high-throughput sequencing (HTS) data but that certain sample characteristics, such as overall target abundance as well as experimental parameters (e.g., bait concentration and secondary structure propensity), consistently influenced enrichment of our diverse set of aDNA samples. Taken together, our results should help guide experimental design, screening strategies, and multiplexed sample equilibration, increasing yield and reducing the expected and actual cost of aDNA EHC high-throughput sequencing projects in the future.

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Section: Resultsmentioning

confidence: 68%

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

2013

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“…The first genetic marker analyzed in human paleogenetic studies was mitochondrial DNA (mtDNA) because of its higher copy number in the cell than nuclear DNA. Probe hybridization assays used biotinylated DNA or RNA probes targeting the two hypervariable segments of the mtDNA control region (CR) ( Briggs et al, 2009 ; Krause et al, 2010 ; Maricic et al, 2010 ; Enk et al, 2013 ; Kihana et al, 2013 ; Templeton et al, 2013 ; Eduardoff et al, 2017 ; Loreille et al, 2018 ). Another uniparental marker, the Y-chromosome DNA (Y-DNA), was also used to study aDNA.…”

Section: Early Developments Of Hybrid-capture Strategies: Human Genetmentioning

confidence: 99%

Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

Gaudin

Desnues

2018

Front. Microbiol.

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“…The rise of MPS-based assays significantly increased the success of obtaining useful genetic data from ancient specimens [ 16 , 17 ], especially by using DNA hybridization enrichment and single-step PCR primer extension capture (PEC) [ 18 ]. Probe hybridization assays use 70–500 base pair (bp)-long biotinylated DNA or RNA probes hybridizing to regions in the mitogenome, and are either generated from PCR products [ 19 , 20 , 21 , 22 ] or designed and made commercially available (e.g., Mybaits (Microarray, Ann Arbor, MI, USA) [ 23 ] or Agilent’s SureSelect (Agilent Technologies, Santa Clara, CA, USA) [ 24 ]). However, the protocols usually require high library DNA input (more than 100 ng), and the hybridization steps can take up to 72 h. Single Step Extension Capture was developed by Briggs et al in 2009 [ 25 ] for the sequence analysis of Neanderthal mitogenomes.…”

Section: Introductionmentioning

confidence: 99%

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

Eduardoff

Xavier

Strobl

et al. 2017

Genes

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The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories.

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Emulsion PCR-coupled target enrichment: An effective fishing method for high-throughput sequencing of poorly preserved ancient DNA

Cited by 19 publications

References 26 publications

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

Quantitative PCR as a Predictor of Aligned Ancient DNA Read Counts Following Targeted Enrichment

Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

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