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A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

ABSTRACT: Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome c oxidase I (COI) gene from 380 butterflies with an average age of 50 years. Nested multiplex PCR was employed for library preparation to facilitate sequence recovery from extracts with low concentrations of highly degraded DNA. By employing circular consensus sequencing (CCS) of short amplicons (circa 150 bp), full-length barcodes could be assembled without a reference sequence, an important advance from earlier protocols which required reference sequences to guide contig assembly. The Sequel protocol recovered COI sequences (499 bp on average) from 318 of 380 specimens (84%), much higher than for Sanger sequencing (26%). Because each read derives from a single molecule, it was also possible to quantify the incidence of substitutions arising from DNA damage. In agreement with past work on sequence changes induced by DNA degradation, the transition C/G ? T/A was the most prevalent category of change, but its rate of occurrence (4.58E-4) was so low that it did not impede the recovery of reliable sequences. Because the current protocol recovers COI sequence from most museum specimens, and because sequence fidelity is unaffected by nucleotide misincorporations, large-scale sequence characterization of museum specimens is feasible.

SUBMITTER: D'Ercole J

PROVIDER: S-EPMC7811786 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

D'Ercole Jacopo J Prosser Sean W J SWJ Hebert Paul D N PDN

PeerJ 20210114

Natural history collections are a valuable resource for molecular taxonomic studies and for examining patterns of evolutionary diversification, particularly in the case of rare or extinct species. However, the recovery of sequence information is often complicated by DNA degradation. This article describes use of the Sequel platform (Pacific Biosciences) to recover the 658 bp barcode region of the mitochondrial cytochrome <i>c</i> oxidase I (COI) gene from 380 butterflies with an average age of 5 ...[more]

PMID: 33520432

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Project description:BackgroundThe degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation ("miscoding lesions") has been the object of extensive investigations.Methodology/principal findingsTo improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350-5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Otzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences.Conclusions/significanceThis study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine-->thymine/guanine-->adenine) transitions and that type 1 transitions are essentially PCR artifacts.

Project description:Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

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Project description:Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R²?=?0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R²?=?0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R²??=?0.96) and whole transcriptome RNA-sequencing following "traditional" library preparation using Illumina NGS kits (R²?=?0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10?-fold range was reduced more than 10,000-fold, from 2.3×10? to 1.4×10? sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

Dataset Information

A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

Publications

A SMRT approach for targeted amplicon sequencing of museum specimens (Lepidoptera)-patterns of nucleotide misincorporation.

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