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

Enk, Jacob; Rouillard, Jean Marie; Poinar, Hendrik N.

doi:10.2144/000114114

Cited by 40 publications

(38 citation statements)

References 48 publications

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“…Unsurprisingly, specimen age and metrics of DNA quantity (pre-library DNA concentration, post-library DNA concentration) and sequence capture rates (raw read count, UCE locus count, UCE mean contig length and UCE reads on target) were negatively correlated. Metrics of DNA concentration were correlated with overall sequencing success, which confirms results from previous studies [11,50]. Pre- and post-library DNA concentrations were a better indicator for UCE contig length than for total UCE locus capture.…”

Section: Discussionsupporting

confidence: 87%

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens

et al. 2016

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Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2–121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6–972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06–9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21–39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens.

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Section: Discussionsupporting

confidence: 87%

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens

et al. 2016

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“…Most laboratories that use NGS libraries for sample screening construct an initial 'test library' for each sample, and then shotgun sequence or enrich it for a small region of interest: a few loci of the genome [3,4], whole mitochondrial DNA (mtDNA) [5,6] or the plasmids of a targeted pathogen [7]. The resulting data can be evaluated for features expected from authentic ancient DNA, such as short molecule sizes, and a high rate of C !…”

Section: Introductionmentioning

confidence: 99%

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

Rohland

Harney

Mallick

et al. 2015

Phil. Trans. R. Soc. B

443

441

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The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.

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“…Sequencing data were curated and aligned, based on the parameters in Enk et al (21), using cutadapt v.1.2 (22), FLASH v.1.0.3 (23), and Burrows-Wheeler Aligner v.0.6.1-r104 (BWA) (24). The mammoth ( Mammuthus primigenius ) mitochondrial genome (GenBank Accession No.…”

Section: Methodsmentioning

confidence: 99%

Surveying the Repair of Ancient DNA from Bones Via High-Throughput Sequencing

et al. 2015

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DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo no longer operate. By incubating degraded DNA with specific enzyme combinations adopted from these pathways, it is possible to reverse some of the post-mortem nucleic acid damage prior to downstream analyses such as library preparation, targeted enrichment, and high-throughput sequencing. Here, we evaluate the performance of two available repair protocols on previously characterized DNA extracts from four mammoths. Both methods use endonucleases and glycosylases along with a DNA polymerase-ligase combination. PreCR Repair Mix increases the number of molecules converted to sequencing libraries, leading to an increase in endogenous content and a decrease in cytosine-to-thymine transitions due to cytosine deamination. However, the effects of Nelson Repair Mix on repair of DNA damage remain inconclusive.

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

Cited by 40 publications

References 48 publications

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

Surveying the Repair of Ancient DNA from Bones Via High-Throughput Sequencing

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