Targeted Capture Approach Research Articles

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

Phylogenomics of Fresh and Formalin Specimens Resolves the Systematics of Old World Mud Snakes (Serpentes: Homalopsidae) and Expands Biogeographic Inference

Our knowledge of the biodiversity of Asia and Australasia continues to expand with more focused studies on systematics of various groups and their biogeography. Historically, fluctuating sea levels and cyclic connection and separation of now-disjunct landmasses have been invoked to explain the accumulation of biodiversity via species pump mechanisms. However, recent research has shown that geological shifts of the mainland and species dispersal events may be better explanations of the biodiversity in these regions. We investigate these processes using the poorly studied and geographically widespread Mud Snakes (Serpentes: Homalopsidae) using a target capture approach of ~4,800 nuclear loci from fresh tissues and supplemental mitochondrial data from formalin tissues from museum specimens. We use these datasets to reconstruct the first resolved phylogeny of the group, identify their biogeographic origins, and test hypotheses regarding the roles of sea-level change and habitat selection on their diversification. Divergence dating and ancestral range estimation yielded support for an Oligocene origin and diversification from mainland Southeast Asia and Sundaland in the rear-fanged group ~20 million years ago, followed by eastward and westward dispersal. GeoHiSSE models indicate that niche expansion of ancestral, rear-fanged lineages into aquatic environments did not impact their diversification rates. Our results highlight that Pleistocene sea-level changes and habitat specificity did not primarily lead to the extant species richness of Homalopsidae and that, alternatively, geological shifts in mainland Southeast Asia may have been a major driver of diversity in this group. We also emphasize the importance of using fresh and degraded tissues, and both nuclear and mitochondrial DNA, for filling knowledge gaps in poorly known but highly diverse and conceptually important groups. Here, Homalopsidae represents a non-traditional but effective model study system for understanding transitions between terrestrial, marine, and freshwater environments.

Selection on standing genetic variation mediates convergent evolution in extremophile fish.

Hydrogen sulfide is a toxic gas that disrupts numerous biological processes, including energy production in the mitochondria, yet fish in the Poecilia mexicana species complex have independently evolved sulfide tolerance several times. Despite clear evidence for convergence at the phenotypic level in these fishes, it is unclear if the repeated evolution of hydrogen sulfide tolerance is the result of similar genomic changes. To address this gap, we used a targeted capture approach to sequence genes associated with sulfide processes and toxicity from five sulfidic and five nonsulfidic populations in the species complex. By comparing sequence variation in candidate genes to a reference set, we identified similar population structure and differentiation, suggesting that patterns of variation in most genes associated with sulfide processes and toxicity are due to demographic history and not selection. But the presence of tree discordance for a subset of genes suggests that several loci are evolving divergently between ecotypes. We identified two differentiation outlier genes that are associated with sulfide detoxification in the mitochondria that have signatures of selection in all five sulfidic populations. Further investigation into these regions identified long, shared haplotypes among sulfidic populations. Together, these results reveal that selection on standing genetic variation in putatively adaptive genes may be driving phenotypic convergence in this species complex.

Drift in the tropics: Phylogenetics and biogeographical patterns in Combretaceae

AbstractAimThe aim of this study was to further advance our understanding of the species‐rich, and ecologically important angiosperm family Combretaceae to provide new insights into their evolutionary history. We assessed phylogenetic relationships in the family using target capture data and produced a dated phylogenetic tree to assess fruit dispersal modes and patterns of distribution.LocationTropical and subtropical regions.Time PeriodCretaceous to present.Major Taxa StudiedFamily Combretaceae is a member of the rosid clade and comprises 10 genera and more than 500 species, predominantly assigned to genera Combretum and Terminalia, and occurring on all continents and in a wide range of ecosystems.MethodsWe use a target capture approach and the Angiosperms353 universal probes to reconstruct a robust dated phylogenetic tree for the family. This phylogenetic framework, combined with seed dispersal traits, biome data and biogeographic ranges, allows the reconstruction of the biogeographical history of the group.ResultsAncestral range reconstructions suggest a Gondwanan origin (Africa/South America), with several intercontinental dispersals within the family and few transitions between biomes. Relative abundance of fruit dispersal types differed by both continent and biome. However, intercontinental colonizations were only significantly enhanced by water dispersal (drift fruit), and there was no evidence that seed dispersal modes influenced biome shifts.Main ConclusionsOur analysis reveals a paradox as drift fruit greatly enhanced dispersal distances at intercontinental scale but did not affect the strong biome conservatism observed.

An initial molecular resolution of the mantellid frogs of the Guibemantis liber complex reveals three new species from northern Madagascar

The small arboreal frog Guibemantis liber (Anura: Mantellidae) has served as an example for the existence of deep conspecific lineages that differ by a substantial amount in mitochondrial DNA but are similar in morphology and bioacoustics and thus are assigned to the same nominal species. During fieldwork in northern Madagascar, we identified additional such lineages and surprisingly, observed close syntopy of two of these at various sites. In-depth study based on DNA sequences of the mitochondrial cytochrome b gene from 338 specimens of G. liber sensu lato from across its range, sequences of four nuclear-encoded markers for 154‒257 of these specimens, a phylogenomic dataset obtained by the FrogCap target capture approach, and additional mitochondrial genes for representatives of most mitochondrial lineages, as well as bioacoustic and morphological comparisons, revealed concordant differentiation among several lineages of the G. liber complex. We identify nine lineages differing by 5.3‒15.5% in cytochrome b and 2.4‒10.1% in the 16S rRNA gene, and find that several of these lack or have only limited allele sharing in the nuclear-encoded genes. Based on sympatric or parapatric occurrence without genetic admixture, combined with differences in bioacoustic and morphological characters, we scientifically name three lineages from northern Madagascar as new species: G. razokysp. nov., G. razandrysp. nov., and G. fotsitendasp. nov. Of these new species, G. razokysp. nov. and G. razandrysp. nov. show widespread syntopy across northern Madagascar and differ in body size and advertisement calls. Guibemantis fotsitendasp. nov. is sister to G. razandrysp. nov., but appears to occur at lower elevations, including in close geographic proximity on the Marojejy Massif. We also detected subtle differences in advertisement calls among various other mitochondrial lineages distributed in the Northern Central East and Southern Central East of Madagascar, but the status and nomenclatural identity of these lineages require further morphological and bioacoustic study of reliably genotyped individuals, and assignment of the three available names in the complex: Rhacophorus liber Peracca, 1893, Gephyromantis albogularis Guibé, 1947, and Gephyromantis variabilis Millot and Guibé, 1951. We discuss the identity and type material of these three nomina, designate a lectotype for Gephyromantis variabilis from Itremo, and flag the collection of new material from their type localities, Andrangoloaka and Itremo, as paramount for a comprehensive revision of the G. liber complex.

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida).

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

Target-capture probes for phylogenomics of the Caenogastropoda.

Target-capture approaches have facilitated a rapid growth in the field of phylogenomics but few probe sets exist for mollusks, an exceptionally rich phylum with unparalleled ecological and morphological diversity. We designed and tested the first universal probe set using Phyluce to capture ultraconserved elements (UCEs) and exon loci from the Subclass Caenogastropoda - one of six major lineages of gastropods. The probe set consists of 29,441 probes designed to target 1,142 UCE loci and 1,933 exon loci (3,075 total). In silico analyses of our probe set yielded an average of 2,110 loci from genomes and 1,389 loci from transcriptomes of diverse caenogastropods, from which an average of 1,669 and 849 loci were retained respectively after screening to remove those that matched multiple contigs. Phylogenetic analyses of the loci extracted from transcriptomes produced well-supported trees very similar to those published based on transcriptomic analyses. Phylogenetic relationships estimated from loci extracted from genomes recover similar phylogenetic relationships, and indicate that the loci targeted with this probe set are informative for resolving deep phylogenetic relationships. An in vitro analysis of the probe set with the Epitoniidae, a diverse caenogastropod family of uncertain affinity and with poorly resolved evolutionary relationships, recovered a total of 2,850 loci. Although preliminary, the analysis of loci captured by our probe set for a small number of epitoniid taxa produced a well-resolved tree indicating that this probe set is also able to resolve relationships at shallower hierarchical scales. Together, the in silico and in vitro analyses indicate that target-capture enrichment with this probe set is a useful tool for reconstructing phylogenetic relationships across taxonomic levels and evolutionary time scales.

Concordance and Discordance in the Phylogenomics of the Wrasses and Parrotfishes (Teleostei: Labridae).

Phylogenomic analysis of large genome-wide sequence data sets can resolve phylogenetic tree topologies for large species groups, help test the accuracy of and improve resolution for earlier multi-locus studies and reveal the level of agreement or concordance within partitions of the genome for various tree topologies. Here we used a target-capture approach to sequence 1088 single-copy exons for more than 200 labrid fishes together with more than 100 outgroup taxa to generate a new data-rich phylogeny for the family Labridae. Our time-calibrated phylogenetic analysis of exon-capture data pushes the root node age of the family Labridae back into the Cretaceous to about 79 Ma years ago. The monotypic Centrogenys vaigiensis, and the order Uranoscopiformes (stargazers) are identified as the sister lineages of Labridae. The phylogenetic relationships among major labrid subfamilies and within these clades were largely congruent with prior analyses of select mitochondrial and nuclear datasets. However, the position of the tribe Cirrhilabrini (fairy and flame wrasses) showed discordance, resolving either as the sister to a crown julidine clade or alternatively sister to a group formed by the labrines, cheilines and scarines. Exploration of this pattern using multiple approaches leads to slightly higher support for this latter hypothesis, highlighting the importance of genome-level data sets for resolving short internodes at key phylogenetic positions in a large, economically important groups of coral reef fishes. More broadly, we demonstrate how accounting for sources of biological variability from incomplete lineage sorting and exploring systematic error at conflicting nodes can aid in evaluating alternative phylogenetic hypotheses. [coral reefs; divergence time estimation; exon-capture; fossil calibration; incomplete lineage sorting.].

Target capture data resolve recalcitrant relationships in the coffee family (Rubioideae, Rubiaceae).

Subfamily Rubioideae is the largest of the main lineages in the coffee family (Rubiaceae), with over 8,000 species and 29 tribes. Phylogenetic relationships among tribes and other major clades within this group of plants are still only partly resolved despite considerable efforts. While previous studies have mainly utilized data from the organellar genomes and nuclear ribosomal DNA, we here use a large number of low-copy nuclear genes obtained via a target capture approach to infer phylogenetic relationships within Rubioideae. We included 101 Rubioideae species representing all but two (the monogeneric tribes Foonchewieae and Aitchinsonieae) of the currently recognized tribes, and all but one non-monogeneric tribe were represented by more than one genus. Using data from the 353 genes targeted with the universal Angiosperms353 probe set we investigated the impact of data type, analytical approach, and potential paralogs on phylogenetic reconstruction. We inferred a robust phylogenetic hypothesis of Rubioideae with the vast majority (or all) nodes being highly supported across all analyses and datasets and few incongruences between the inferred topologies. The results were similar to those of previous studies but novel relationships were also identified. We found that supercontigs [coding sequence (CDS) + non-coding sequence] clearly outperformed CDS data in levels of support and gene tree congruence. The full datasets (353 genes) outperformed the datasets with potentially paralogous genes removed (186 genes) in levels of support but increased gene tree incongruence slightly. The pattern of gene tree conflict at short internal branches were often consistent with high levels of incomplete lineage sorting (ILS) due to rapid speciation in the group. While concatenation- and coalescence-based trees mainly agreed, the observed phylogenetic discordance between the two approaches may be best explained by their differences in accounting for ILS. The use of target capture data greatly improved our confidence and understanding of the Rubioideae phylogeny, highlighted by the increased support for previously uncertain relationships and the increased possibility to explore sources of underlying phylogenetic discordance.

Capturing the antibiotic resistome of preterm infants reveals new benefits of probiotic supplementation

BackgroundProbiotic use in preterm infants can mitigate the impact of antibiotic exposure and reduce rates of certain illnesses; however, the benefit on the gut resistome, the collection of antibiotic resistance genes, requires further investigation. We hypothesized that probiotic supplementation of early preterm infants (born < 32-week gestation) while in hospital reduces the prevalence of antibiotic resistance genes associated with pathogenic bacteria in the gut. We used a targeted capture approach to compare the resistome from stool samples collected at the term corrected age of 40 weeks for two groups of preterm infants (those that routinely received a multi-strain probiotic during hospitalization and those that did not) with samples from full-term infants at 10 days of age to identify if preterm birth or probiotic supplementation impacted the resistome. We also compared the two groups of preterm infants up to 5 months of age to identify persistent antibiotic resistance genes.ResultsAt the term corrected age, or 10 days of age for the full-term infants, we found over 80 antibiotic resistance genes in the preterm infants that did not receive probiotics that were not identified in either the full-term or probiotic-supplemented preterm infants. More genes associated with antibiotic inactivation mechanisms were identified in preterm infants unexposed to probiotics at this collection time-point compared to the other infants. We further linked these genes to mobile genetic elements and Enterobacteriaceae, which were also abundant in their gut microbiomes. Various genes associated with aminoglycoside and beta-lactam resistance, commonly found in pathogenic bacteria, were retained for up to 5 months in the preterm infants that did not receive probiotics.ConclusionsThis pilot survey of preterm infants shows that probiotics administered after preterm birth during hospitalization reduced the diversity and prevented persistence of antibiotic resistance genes in the gut microbiome. The benefits of probiotic use on the microbiome and the resistome should be further explored in larger groups of infants. Due to its high sensitivity and lower sequencing cost, our targeted capture approach can facilitate these surveys to further address the implications of resistance genes persisting into infancy without the need for large-scale metagenomic sequencing.CuDbLCLZC-DiSQEiNZt-Y2Video

A targeted capture approach to generating reference sequence databases for chloroplast gene regions.

Metabarcoding has improved the way we understand plants within our environment, from their ecology and conservation to invasive species management. The notion of identifying plant taxa within environmental samples relies on the ability to match unknown sequences to known reference libraries. Without comprehensive reference databases, species can go undetected or be incorrectly assigned, leading to false‐positive and false‐negative detections. To improve our ability to generate reference sequence databases, we developed a targeted capture approach using the OZBaits_CP V1.0 set, designed to capture chloroplast gene regions across the entirety of flowering plant diversity. We focused on generating a reference database for coastal temperate plant species given the lack of reference sequences for these taxa. Our approach was successful across all specimens with a target gene recovery rate of 92%, which was achieved in a single assay (i.e., samples were pooled), thus making this approach much faster and more efficient than standard barcoding. Further testing of this database highlighted 80% of all samples could be discriminated to family level across all gene regions with some genes achieving greater resolution than others—which was also dependent on the taxon of interest. Thus, we demonstrate the importance of generating reference sequences across multiple chloroplast gene regions as no single loci are sufficient to discriminate across all plant groups. The targeted capture approach outlined in this study provides a way forward to achieve this.

A 638‐gene phylogeny supports the recognition of twice as many species in the Malagasy endemic genusCapurodendron(Sapotaceae)

AbstractThe Malagasy genusCapurodendroncurrently accommodates 26 described species and is the largest genus of the family Sapotaceae in Madagascar. These species are frequently logged because of their valued hardwood, which potentially puts them at risk of extinction. Species‐level identifications are often problematic, and this hinders both an accurate assessment of their conservation status and the development of effective protection measures. We sorted all the material (ca. 860 collections) available in the herbaria with significant collections for Madagascar into 47 putative species based on morphology. On 41 of these, for which we were able to retrieve suitable DNA, we conducted a phylogenetic reconstruction based on molecular sequences of 638 loci from 108Capurodendronspecimens, performing a target capture approach combined with next‐generation sequencing. Maximum likelihood (RAxML), pseudocoalescence (ASTRAL), and coalescence (STACEY) analyses showed thatCapurodendroncomprises two deeply divergent lineages. One, which includes a single species, is here newly described asC. subg.Reflexisepalabased on its distinctive morphology. The second lineage contains all remaining species, which seem to have resulted from a rapid radiation event. The phylogenetic tree provides good support for most of the species hypothesized based on morphology, with the exception of two species‐groups that we have named the Arid Complex and the Eastern Complex. As many as 20 species‐level lineages genetically distinct from any of the currently recognized species were identified, 17 of which were morphologically well‐characterized, representing strong candidates for new species. This would suggest thatCapurodendronis the most species‐rich endemic genus of plants in Madagascar. While 14 of these 20 clades are still under study, we here describe six species new to science:Capurodendron andrafiamenae(provisionally assessed as CR),C. aubrevillei(VU),C. birkinshawii(CR),C. naciriae(EN),C. randrianaivoi(CR), andC. sakarivorum(EN).Capurodendron oblongifoliumcomb. nov. (EN), previously regarded as a variety ofC. perrieri, represents a distinct lineage that is here recognized at the species level. The newly described species are illustrated by line drawings and photographs from the field, and a preliminary threat assessment is provided. We discuss the evolutionary history ofCapurodendronand also explore the question of node age estimates and their methodological limitations.

Systematics and biogeography of the whistlers (Aves: Pachycephalidae) inferred from ultraconserved elements and ancestral area reconstruction

The utility of islands as natural laboratories of evolution is exemplified in the patterns of differentiation in widespread, phenotypically variable lineages. The whistlers (Aves: Pachycephalidae) are one of the most complex avian radiations, with a combination of widespread and locally endemic taxa spanning the vast archipelagos of the Indo-Pacific, making them an ideal group to study patterns and processes of diversification on islands. Here, we present a robust, species-level phylogeny of all five genera and 85% of species within Pachycephalidae, based on thousands of ultraconserved elements (UCEs) generated with a target-capture approach and high-throughput sequencing. We clarify phylogenetic relationships within Pachycephala and report on divergence timing and ancestral range estimation. We explored multiple biogeographic coding schemes that incorporated geological uncertainty in this complex region. The biogeographic origin of this group was difficult to discern, likely owing to aspects of dynamic Earth history in the Indo-Pacific. The Australo-Papuan region was the likely origin of crown-group whistlers, but the specific ancestral area could not be identified more precisely than Australia or New Guinea, and Wallacea may have played a larger role than previously realized in the evolutionary history of whistlers. Multiple independent colonizations of island archipelagos across Melanesia, Wallacea, and the Philippines contributed to the relatively high species richness of extant whistlers. This work refines our understanding of one of the regions’ most celebrated bird lineages and adds to our growing knowledge about the patterns and processes of diversification in the Indo-Pacific.

TheSticta filix - Sticta laceraconundrum (lichenized Ascomycota: Peltigeraceae subfamily Lobarioideae): unresolved lineage sorting or developmental switch?

AbstractWe assessed the status of two New Zealand endemic morphodemes in the genus Sticta, currently treated as two separate taxa, Sticta filix and Sticta lacera. Both are green-algal lichens with a distinct stipe that grow in forested habitats and are suitable indicators of the indigenous vegetation health in forest ecosystems in New Zealand. They exhibit different morphologies and substrate ecologies: S. filix forms rather robust thalli, often on exposed trunks of phorophytes, with erect stems distinctly emerging from the substrate, whereas S. lacera is a more delicate lichen growing near the base of trees, usually among bryophyte mats or sheltered in the exposed portions of the phorophyte root-plate, with a prostrate, branched, stolon-like stem barely emerging from the substrate. Throughout their range, both taxa grow sympatrically and often in close proximity (syntopically). Despite the differences, ITS barcoding does not support the two morphodemes as separate species. In this study we assessed two possible explanations: (1) S. filix and S. lacera are discrete phenotypes of a single species, caused by developmental switching triggered by a discrete environmental variable, the propagules developing either on bare substrate or between bryophytes; and (2) the two morphodemes represent separate lineages, but ITS does not provide sufficient resolution to reflect this. We performed a quantitative analysis of morphological and ecological parameters, based on vouchered herbarium collections and field observations on iNaturalist NZ (https://inaturalist.nz), to assess the level of discreteness of the growth forms and to test for a correlation with the presence of a bryophyte mat. We further took advantage of an existing molecular data set from a target capture approach, comprised of 205 protein markers. This data set was used to establish a framework of percentage identities between pairs of the same and of different species among lobarioid Peltigeraceae and then to test whether the S. filix/lacera pairing fell closer to a within-species or a between-species pairing. The morphometric analysis of herbarium material resolved S. filix and S. lacera as two discrete morphs with little overlap, supported by numerous observations on iNaturalist NZ. However, whereas herbarium material suggested a significant association of the lacera morph with bryophyte mats, no such pattern was evident from field images on iNaturalist NZ, in which both morphs frequently associated with bryophyte mats. This highlights the limitations of herbarium material to correctly assess substrate ecology, whereas iNaturalist NZ postings had issues with correct identifications, given that especially S. lacera is easily confused with Pseudocyphellaria multifida. Based on the target capture data, the percentage identity of the S. filix/lacera pairing (99.43%) was significantly higher than that of all 12 between-species pairings (93.20–98.01%); it was at the same time lower than that of all within-species pairings (99.63–99.99%) but significantly so only in comparison with five out of the eight within-species pairings. The target capture approach is thus inconclusive, but the combination of all data suggests that S. filix and S. lacera are not discrete morphodemes of a single species but represent two separate lineages which emerged recently and hence cannot be resolved using the ITS barcoding marker or even a deeper phylogenomic approach based on protein-coding markers. We propose transplantation experiments and the application of RADseq to further assess this situation.

A Multi-Gene Region Targeted Capture Approach to Detect Plant DNA in Environmental Samples: A Case Study From Coastal Environments

Metabarcoding of plant DNA recovered from environmental samples, termed environmental DNA (eDNA), has been used to detect invasive species, track biodiversity changes, and reconstruct past ecosystems. The P6 loop of the trnL intron is the most widely utilised gene region for metabarcoding plants due to the short fragment length and subsequent ease of recovery from degraded DNA, which is characteristic of environmental samples. However, the taxonomic resolution for this gene region is limited, often precluding species level identification. Additionally, targeting gene regions using universal primers can bias results as some taxa will amplify more effectively than others. To increase the ability of DNA metabarcoding to better resolve flowering plant species (angiosperms) within environmental samples, and reduce bias in amplification, we developed a multi-gene targeted capture method that simultaneously targets 20 chloroplast gene regions in a single assay across all flowering plant species. Using this approach, we effectively recovered multiple chloroplast gene regions for three species within artificial DNA mixtures down to 0.001 ng/μL of DNA. We tested the detection level of this approach, successfully recovering target genes for 10 flowering plant species. Finally, we applied this approach to sediment samples containing unknown compositions of eDNA and confidently detected plant species that were later verified with observation data. Targeting multiple chloroplast gene regions in environmental samples, enabled species-level information to be recovered from complex DNA mixtures. Thus, the method developed here, confers an improved level of data on community composition, which can be used to better understand flowering plant assemblages in environmental samples.

Recurrent mutations in multiple components of the SWI/SNF complex in myelodysplastic syndromes and acute myeloid leukaemia

Recurrent mutations in multiple components of the SWI/SNF complex in myelodysplastic syndromes and acute myeloid leukaemia

Genomic library preparation and hybridization capture of formalin-fixed tissues and allozyme supernatant for population genomics and considerations for combining capture- and RADseq-based single nucleotide polymorphism data sets.

Until recently many historical museum specimens were largely inaccessible to genomic inquiry, but high-throughput sequencing (HTS) approaches have allowed researchers to successfully sequence genomic DNA from dried and fluid-preserved museum specimens. In addition to preserved specimens, many museums contain large series of allozyme supernatant samples, but the amenability of these samples to HTS has not yet been assessed. Here, we compared the performance of a target-capture approach using alternative sources of genomic DNA from 10 specimens of spring salamanders (Plethodontidae: Gyrinophilus porphyriticus) collected between 1985 and 1990: allozyme supernatants, allozyme homogenate pellets and formalin-fixed tissues. We designed capture probes based on double-digest restriction-site associated sequencing (RADseq) derived loci from frozen blood samples available for seven of the specimens and assessed the success and consistency of capture and RADseq approaches. This study design enabled direct comparisons of data quality and potential biases among the different data sets for phylogenomic and population genomic analyses. We found that in phylogenetic analyses, all enrichment types for a given specimen clustered together. In principal component space all capture-based samples clustered together, but RADseq samples did not cluster with corresponding capture-based samples. Single nucleotide polymorphism calls were on average 18.3% different between enrichment types for a given individual, but these discrepancies were primarily due to differences in heterozygous/homozygous single nucleotide polymorphism calls. We demonstrate that both allozyme supernatant and formalin-fixed samples can be successfully used for population genomic analyses and we discuss ways to identify and reduce biases associated with combining capture and RADseq data.

Target capture sequencing of SARS-CoV-2 genomes using the ONETest Coronaviruses Plus

We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence the SARS-CoV-2 genome and select loci of other respiratory viruses. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a PCR assay. For 48 of the samples, we also applied the ARTIC protocol. Of the 70 ONETest libraries, 45 (64%) had a (near-)complete sequence (>29,000 bases and >90% covered by >9 reads). Of the 48 ARTIC libraries, 25 (52%) had a (near-)complete sequence. In 19 out of 25 (76%) samples in which both the ONETest and ARTIC yielded (near-)complete sequences, the lineages assigned were identical. As a target capture approach, the ONETest is less prone to loss of sequence coverage than amplicon approaches, and thus can provide complete genomic information more often to track and monitor SARS-CoV-2 variants.

Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens.

Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single-stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step-by-step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies.