Tagged Amplicon Research Articles

Monitoring Bacterial Community Dynamics in Abalone (Haliotis discus hannai) and the Correlations Associated with Aquatic Diseases

Bacteria are an integral component of their host. However, information about the microbiota living in and around many aquatic animals is lacking. In this study, multiplex bar-coded pyrosequencing of the 16S ribosomal RNA gene was used to monitor the dynamics of abalone, Haliotis discus hannai, bacterial communities in the intestine, water from cement culture ponds, and surrounding sea areas. Correlations between the bacterial communities and common aquaculture diseases were also evaluated. A total of 329,798 valid sequences and 15,277 operational taxonomic units (OTUs) from 32 samples were obtained by 454 tag amplicon pyrosequencing. The Shannon indices of the seawater samples ranged from 2.84 to 5.6 and the Shannon indices of the abalone intestine samples ranged from 1.2 to 5.12, which were much lower than those of seawater. The dominant phyla in seawater samples were Proteobacteria, Bacteroidetes, Fusobacteria, Cyanobacteria, etc. The dominant phyla in the abalone intestine varied greatly in different months. The dominant genera in the seawater of the cement culture ponds changed in different months, mainly Psychrilyobacter and Pseudoalteromonas. The dominant genera in seawater from the open sea vary considerably between months. The dominant genus of bacteria in the abalone intestine during the months when abalones are susceptible to disease is mainly Mycoplasma spp. Canonical correspondence analysis revealed that bacterial communities in seawater and the intestine responded differently to environmental variables, with similar microbiota in the same area. pH, dissolved oxygen concentration, and temperature were closely related to the samples from the sea area. Oxidation-reduction potential, salinity, phosphate, nitrate, and ammonia nitrogen concentrations were closely related to the water samples from the artificial pools. These findings may add significantly to our understanding of the complex interactions between microbiota and environmental variables in the abalone intestine as well as in the surrounding seawater.

Next-generation phylogeography of the banded newts (Ommatotriton): A phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure

Technological developments now make it possible to employ many markers for many individuals in a phylogeographic setting, even for taxa with large and complex genomes such as salamanders. The banded newt (genus Ommatotriton) from the Near East has been proposed to contain three species (O. nesterovi, O. ophryticus and O. vittatus) with unclear phylogenetic relationships, apparently limited interspecific gene flow and deep intraspecific geographic mtDNA structure. We use parallel tagged amplicon sequencing to obtain 177 nuclear DNA markers for 35 banded newts sampled throughout the range. We determine population structure (with Bayesian clustering and principal component analysis), interspecific gene flow (by determining the distribution of species-diagnostic alleles) and phylogenetic relationships (by maximum likelihood inference of concatenated sequence data and based on a summary-coalescent approach). We confirm that the three proposed species are genetically distinct. A sister relationship between O. nesterovi and O. ophryticus is suggested. We find evidence for introgression between O. nesterovi and O. ophryticus, but this is geographically limited. Intraspecific structuring is extensive, with the only recognized banded newt subspecies, O. vittatus cilicensis, representing the most distinct lineage below the species level. While mtDNA mostly mirrors the pattern observed in nuclear DNA, all banded newt species show mito-nuclear discordance as well.

Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay in place

Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay in place Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of ecological interactions, diet and biodiversity, as its labelling of amplicons allows high-throughput sequencing of taxonomically informative DNA sequences from many samples in parallel. The backbone of metabarcoding is the addition of sample-specific nucleotide identifiers to amplicons and then following sequencing using these to assign metabarcoding sequences to the samples they originated from. This allows the pooling of hundreds to thousands of samples before sequencing and thereby full utilisation of the capacity of high-throughput sequencing platforms. The nucleotide identifiers can be added both during the metabarcoding PCR and during library preparation, i.e. when amplicons are prepared for sequencing. There are three main strategies with which to achieve nucleotide labelling in metabarcoding studies. One commonly used strategy is the so-called tagged PCR approach in which DNA extracts are individually amplified with metabarcoding primers that carry sample-specific nucleotide tags at the 5’ end. The uniquely tagged products are then pooled and a library prepared on the pool of amplicons. However, tag‐jumps have been documented in this commonly used metabarcoding approach (Schnell et al. 2015). Tag-jumps cause nucleotide tags to switch between amplicons, resulting in occurrence of amplicons that carry different tags than originally applied. Sequences in the sequencing output that carry tag combinations not used in the study design are easily identified and excluded. However, sequences carrying incorrect, but already used, tag combinations will cause incorrect assignments of sequences to samples. This can - much to the detriment of metabarcoding studies - lead to false positives and artificial inflation of diversity in the samples (Schnell et al. 2015). The occurrence of tag-jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin-tags to ensure that tag‐jumps cannot result in false assignments of sequences to samples (Schnell et al. 2015). However, this increases both cost and workload of metabarcoding studies. In a recently published article, we demonstrate a tag-jump free single-tube library preparation protocol for Illumina sequencing specifically designed for 5’ nucleotide tagged amplicons, the Tagsteady protocol (Carøe & Bohmann 2020). We designed the Tagsteady protocol to circumvent the two steps during library preparation of pools of 5ʹ nucleotide-tagged amplicons that had previously been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step, and ii) post-ligation PCR amplification of amplicon libraries. We used pools of twin‐tagged amplicons to investigate the effect of these two steps on the occurrence of tag‐jumps. Doing this, we demonstrated that blunt‐ending and post-ligation PCR, alone or together, can result in high proportions of tag-jumps, in our study up to ca. 49% of total sequences. The Tagsteady protocol where both these steps were left out resulted in tag‐jump levels comparable to background contamination (Carøe & Bohmann 2020). In our study, we encourage practitioners to avoid using T4 DNA polymerase blunt‐ending and post-ligation PCR in library preparation of 5’ nucleotide tagged amplicon pools, for example by using the Tagsteady protocol (Carøe & Bohmann 2020). This will enable efficient and cost-effective generation of metabarcoding data with correct assignment of sequences to samples. References Carøe C, Bohmann K (2020) Tagsteady: A metabarcoding library preparation protocol to avoid false assignment of sequences to samples. Molecular Ecology Resources, 20, 1620–1631. Schnell IB, Bohmann K, Gilbert MTP (2015) Tag jumps illuminated - reducing sequence-to-sample misidentifications in metabarcoding studies. Molecular Ecology Resources, 15, 1289–1303.

An unusual microbiome characterises a spatially-aggressive crustose alga rapidly overgrowing shallow Caribbean reefs

Several species of crustose coralline algae (CCA) and their associated microbial biofilms play important roles in determining the settlement location of scleractinian corals on tropical reefs. In recent decades, peyssonnelid algal crusts (PAC) have become spatial dominants across large areas of shallow Caribbean reefs, where they appear to deter the recruitment of scleractinians. Our genetic investigations of PAC in St. John, US Virgin Islands, amplifying the large-subunit ribosomal RNA and psbA protein D1 marker genes, revealed them to be identical to Ramicrusta textilis previously reported overgrowing corals in Jamaica. Specimens of PAC sampled from the Honduras were likewise identical, confirming that this crustose alga inhabits the easternmost and westernmost regions of the Caribbean. We also analysed 16S rDNA tag amplicon libraries of the biofilms associated with PAC and sympatric CCA, which is favoured for coral settlement. Our results show that the microbial communities on PAC (vs. CCA) are characterized by significantly lower numbers of the epibiotic bacterial genus Pseudoalteromonas, which facilitates the recruitment and settlement of marine invertebrates. From these data, we infer that PAC are therefore unlikely to be attractive as settlement sites for coral larvae. Given the significant ecological change anticipated on these reefs due to increasing cover of PAC, there is an urgent need to further investigate competitive interactions between PAC and scleractinian corals, and elucidate the role of PAC and their associated microbiomes in accentuating phase shifts from coral to algae on tropical reefs.

Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in the JAK3 gene.

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring “driver” genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency.Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.

Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

Targeted sequencing of plasma cell-free DNA to predict response to PD1 inhibitors in advanced non-small cell lung cancer.

103 Background: Tumor mutational burden is an emerging biomarker of response to immune checkpoint inhibitors (ICI), whose clinical adoption is challenging, especially in liquid biopsies. We hypothesized that targeting limited but relevant genetic alterations in plasma cell-free DNA with next generation sequencing (NGS) along with early monitoring may represent a non-invasive approach to predict response to ICI. Methods: Plasma samples from responders (PFS > 6 months) and non-responders (progressive disease at first evaluation) patients collected before nivolumab (second line) initiation and at 1 month were tested using tagged amplicon sequencing of hotspots and coding regions from 36 genes, blinded to clinical outcomes and tumor genotype. Molecular profile of ctDNA, and its early kinetics (1 month) were analyzed as potential early indicators of response. Results: 98 patients were analyzed, of which 86 (39 responders, 47 non-responders) were evaluable for response. Alterations in ctDNA were detectable in 67/86 baseline samples (78%). The detection of a targetable oncogenic driver (5 EGFR, 1 ALK) was associated with progressive disease on the first CT-scan The presence of a PTEN and/or STK11 mutations (b-PS(+)) was correlated with poor outcomes (median PFS 1.5 months vs. 8 months in b-PS(-)) patients, p = 0.0007), while the presence of transversion mutations in KRAS and/or TP53 (b-KP-Tv(+)) predicted good outcomes (median PFS 11 months vs. 2 months in b-KP-Tv(-) patients, p = 0.0088). Combining these results, patients with a low “immune score” (driver and/or b-PS(+) and/or b-KP-Tv(-)) derived poor outcomes (PFS 2 months), compared with patients with a high immune score (no driver, b-PS(-) and b-KP-Tv(+), PFS 14 months, p = 0.0001, HR 2.96). Studying early changes in 65 specimens, molecular response was correlated with clinical outcomes (14 months PFS in patients with early ctDNA decrease compared to 2 months in patients with increase, p < 0.0001; HR 2.7). Using cut-off of 30% and 50% of plasma response increased the ability of ctDNA to predict response (HR 4 and 4.17, respectively). Conclusions: Targeted sequencing of plasma ctDNA and its early variations can predict response to anti-PD-1. Clinical trial information: NCT02827344.

Abstract 939: Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients

Abstract Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA. The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots. The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965). This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients. Citation Format: Samuel Woodhouse, Vincent Plagnol, Karen Howarth, Stefanie Lensing, Matt Smith, Michael Epstein, Mikidache Madi, Sarah Smalley, Catherine Leroy, Jonathan Hinton, Frank de Kievit, Esther Musgrave-Brown, Colin Herd, Katherine Baker-Neblett, Will Brennan, Peter Dimitrov, Nathan Campbell, Nitzan Rosenfeld, James Clark, Davina Gale, Jamie Platt, John Calaway, Greg Jones, Tim Forshew. Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 939.

A MinION™-based pipeline for fast and cost-effective DNA barcoding.

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory and is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION™ reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION™ barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION™ barcodes have an accuracy of 99.3%-100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be <USD 2.

Towards holomorphology in entomology: rapid and cost‐effective adult–larva matching using NGS barcodes

AbstractIn many taxa the morphology of females and immatures is poorly known because species descriptions and identification tools have a male bias. The root causes are problems with matching life‐history stages and genders belonging to the same species. Such matching is time‐consuming when conventional methods are used (e.g. rearing) and expensive when the stages are matched with DNA barcodes. Unfortunately, the lack of associations is not a trivial problem because it renders a large part of the phenome of insects unexplored, although larvae and females are useful sources of characters for descriptive and phylogenetic purposes. In addition, many collectors intentionally avoid females and immature stages, which skews survey results, interferes with collecting life‐history information, and makes it less likely that rare species are discovered. These problems even exist for well‐studied taxa like Odonata, where obtaining adult–larva matches relies largely on rearing. Here we demonstrate how the matching problem can be addressed with cost‐effective tagged amplicon sequencing of a 313‐bp segment of cox1 with next‐generation sequencing (NGS) (‘NGS barcoding’). We illustrate the value of this approach based on Singapore's odonate fauna which is of a similar size as the European fauna (Singapore, 122 extant species; Europe, 138 recorded species). We match the larvae and adults of 59 species by first creating a barcode database for 338 identified adult specimens representing 83 species. We then sequence 1178 larvae from a wide range of sources. We successfully barcode 1123 specimens, which leads to adult–larva matches for 59 species based on our own barcodes (55) and online barcode databases (4). With these additions, 84 of the 131 species recorded in Singapore have now been associated with a species name. Most common species are now matched (83%), and good progress has been made for vulnerable/near‐threatened (55%), endangered (53%), and critically endangered species (38%). We used nondestructive DNA extraction methods in order to be able to use high‐resolution imaging of matched larvae to establish a publicly available digital reference collection for odonates which is incorporated into ‘Biodiversity of Singapore’ ( https://singapore.biodiversity.online/). We suggest that the methods described here are suitable for many insect taxa because NGS barcoding allows for fast and low‐cost matching of well‐studied life‐history stages with neglected semaphoronts (eggs, larvae, females). We estimate that the specimen‐specific amplicons in this study (c. 1500 specimens) can now be obtained within eight working days and that the laboratory and sequencing cost is c. US$600 (&lt; US$0.40 per specimen).

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer

BackgroundGenomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methodsPlasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. ResultsA total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2=0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. ConclusionsThis blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

Early detection of competing resistance mutations using plasma next-generation sequencing (NGS) in patients (pts) with EGFR-mutant NSCLC treated with osimertinib.

11529 Background: In pts with EGFR+ NSCLC, genotyping of plasma cell-free DNA (cfDNA) has become a routine option for non-invasive detection of EGFRT790M. We hypothesized that serial NGS of cfDNA would allow early detection of co-existent resistance mutations during osimertinib treatment. Methods: Serial plasma samples were collected from pts with advanced EGFR+ NSCLC and T790M+ acquired resistance treated with osimertinib. Up to 4 specimens were analyzed, blinded to tumor genotype: baseline, initial 2 follow-ups, and progression. Plasma NGS was performed using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes. Diagnostic accuracy was compared to tumor genotype (including NGS when available) and droplet digital PCR (ddPCR) of cfDNA. Results: 94 specimens from 26 pts underwent plasma NGS. Studying 26 baseline specimens, plasma NGS was more sensitive than ddPCR for known EGFR driver mutations (100% vs 88.5%). In 8 pts with pretreatment tumor NGS, 5 of 6 TP53 mutations were detected; one plasma positive/tissue negative result was seen across 36 genes, a PIK3CA mutation (0.6% AF) which was confirmed using ddPCR (99.6% specificity). Quantitative concordance of AF compared to ddPCR was high (R = 0.94). 21 pts had detectable EGFR driver mutations at resistance. Among 6 pts with maintained T790M, 4 acquired C797S and 2 of these additionally acquired low level KRAS mutations (G13D, Q61K). Among 15 pts with loss of T790M, 7 had competing non- EGFR alterations identified: MET amp, PIK3CA E545K, BRAF V600E (n = 2), HER2 amp, KRAS G12S, and FGFR1 amp. Resistance mutations detected at AF &gt; 0.3% were confirmed with ddPCR. In 3 pts, a competing resistance mutation (1 KRAS, 2 BRAF) could be detected in plasma NGS pretreatment and reemerged as putative drivers at time of resistance. Conclusions: In this retrospective blinded validation, tagged amplicon-based plasma NGS was more sensitive than ddPCR with high specificity and quantitative concordance. In a subset of cases, serial plasma NGS can detect emergence of competing resistance mutations, creating an opportunity for the study of osimertinib-based targeted therapy combinations.

POSEIDON trial phase 1b results: Safety and preliminary efficacy of the isoform selective PI3K inhibitor taselisib (GDC-0032) combined with tamoxifen in hormone receptor (HR) positive, HER2-negative metastatic breast cancer (MBC) patients (pts) - including response monitoring by plasma circulating tumor (ct) DNA.

2520Background: Endocrine therapy resistance remains a major challenge in the treatment of HR-positive breast cancer. The strategy of combining PI3K-AKT-mTOR pathway inhibitors with endocrine therapy can improve clinical outcomes (eg. BOLERO-2, BELLE-2) but at the cost of increased toxicity. Taselisib is a potent, selective, beta-isoform sparing inhibitor of PI3K designed to have a better therapeutic index than first-generation pan-PI3K inhibitors. Methods: Pts with prior exposure to endocrine therapy were enrolled using a 3+3 design, testing taselisib in combination with tamoxifen 20mg qd. A (21 day on / 7 day off) intermittent schedule was also compared with daily continuous dosing (28/28). The primary objective was to determine the recommended phase II dose and schedule. Other objectives included: pharmacokinetics (PK), pharmacodynamics (PD) and serial monitoring of plasma ctDNA using digital PCR / tagged amplicon deep-sequencing. Results: 30 pts were enrolled in 3 cohorts: taselisib 2mg (21/7), 4mg (2...

Effects of dietary linseed oil and propionate precursors on ruminal microbial community, composition, and diversity in Yanbian yellow cattle.

The rumen microbial ecosystem is a complex system where rumen fermentation processes involve interactions among microorganisms. There are important relationships between diet and the ruminal bacterial composition. Thus, we investigated the ruminal fermentation characteristics and compared ruminal bacterial communities using tag amplicon pyrosequencing analysis in Yanbian yellow steers, which were fed linseed oil (LO) and propionate precursors. We used eight ruminally cannulated Yanbian yellow steers (510 ± 5.8 kg) in a replicated 4 × 4 Latin square design with four dietary treatments. Steers were fed a basal diet that comprised 80% concentrate and 20% rice straw (DM basis, CON). The CON diet was supplemented with LO at 4%. The LO diet was also supplemented with 2% dl-malate or 2% fumarate as ruminal precursors of propionate. Dietary supplementation with LO and propionate precursors increased ruminal pH, total volatile fatty acid concentrations, and the molar proportion of propionate. The most abundant bacterial operational taxonomic units in the rumen were related to dietary treatments. Bacteroidetes dominated the ruminal bacterial community and the genus Prevotella was highly represented when steers were fed LO plus propionate precursors. However, with the CON and LO diet plus malate or fumarate, Firmicutes was the most abundant phylum and the genus Ruminococcus was predominant. In summary, supplementing the diets of ruminants with a moderate level of LO plus propionate precursors modified the ruminal fermentation pattern. The most positive responses to LO and propionate precursors supplementation were in the phyla Bacteriodetes and Firmicutes, and in the genus Ruminococcus and Prevotella. Thus, diets containing LO plus malate or fumarate have significant effects on the composition of the rumen microbial community.

Parallel tagged amplicon sequencing of transcriptome‐based genetic markers for Triturus newts with the Ion Torrent next‐generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing

The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in *BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms.

Genetic analyses of bolting in bulb onion (Allium cepa L.)

We present the first evidence for a QTL conditioning an adaptive trait in bulb onion, and the first linkage and population genetics analyses of candidate genes involved in photoperiod and vernalization physiology. Economic production of bulb onion (Allium cepa L.) requires adaptation to photoperiod and temperature such that a bulb is formed in the first year and a flowering umbel in the second. 'Bolting', or premature flowering before bulb maturation, is an undesirable trait strongly selected against by breeders during adaptation of germplasm. To identify genome regions associated with adaptive traits we conducted linkage mapping and population genetic analyses of candidate genes, and QTL analysis of bolting using a low-density linkage map. We performed tagged amplicon sequencing of ten candidate genes, including the FT-like gene family, in eight diverse populations to identify polymorphisms and seek evidence of differentiation. Low nucleotide diversity and negative estimates of Tajima's D were observed for most genes, consistent with purifying selection. Significant population differentiation was observed only in AcFT2 and AcSOC1. Selective genotyping in a large 'Nasik Red × CUDH2150' F2 family revealed genome regions on chromosomes 1, 3 and 6 associated (LOD > 3) with bolting. Validation genotyping of two F2 families grown in two environments confirmed that a QTL on chromosome 1, which we designate AcBlt1, consistently conditions bolting susceptibility in this cross. The chromosome 3 region, which coincides with a functionally characterised acid invertase, was not associated with bolting in other environments, but showed significant association with bulb sucrose content in this and other mapping pedigrees. These putative QTL and candidate genes were placed on the onion map, enabling future comparative studies of adaptive traits.

Parallel tagged next-generation sequencing on pooled samples - a new approach for population genetics in ecology and conservation.

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches – parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (FST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.

A fast, highly sensitive double‐nested PCR‐based method to screen fish immunobiomes

Efficient methods for constructing 16S tag amplicon libraries for pyrosequencing are needed for the rapid and thorough screening of infectious bacterial diversity from host tissue samples. Here we have developed a double-nested PCR methodology that generates 16S tag amplicon libraries from very small amounts of bacteria/host samples. This methodology was tested for 133 kidney samples from the lake whitefish Coregonus clupeaformis (Salmonidae) sampled in five different lake populations. The double-nested PCR efficiency was compared with two other PCR strategies: single primer pair amplification and simple nested PCR. The double-nested PCR was the only amplification strategy to provide highly specific amplification of bacterial DNA. The resulting 16S amplicon libraries were synthesized and pyrosequenced using 454 FLX technology to analyse the variation of pathogenic bacteria abundance. The proportion of the community sequenced was very high (Good's coverage estimator; mean = 95.4%). Furthermore, there were no significant differences of sequence coverage among samples. Finally, the occurrence of chimeric amplicons was very low. Therefore, the double-nested PCR approach provides a rapid, informative and cost-effective method for screening fish immunobiomes and most likely applicable to other low-density microbiomes as well.