Hybrid Capture Method Research Articles

Profiling T-Cell Receptor Diversity and Dynamics during Lymphoma Immunotherapy Using Cell-Free DNA (cfDNA)

Profiling T-Cell Receptor Diversity and Dynamics during Lymphoma Immunotherapy Using Cell-Free DNA (cfDNA)

Dynamics of severe acute respiratory syndrome coronavirus 2 genome variants in the feces during convalescence

In response to the current coronavirus disease 2019 (COVID-19) pandemic, it is crucial to understand the origin, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which relies on close surveillance of genomic diversity in clinical samples. Although the mutation at the population level had been extensively investigated, how the mutations evolve at the individual level is largely unknown. Eighteen time-series fecal samples were collected from nine patients with COVID-19 during the convalescent phase. The nucleic acids of SARS-CoV-2 were enriched by the hybrid capture method. First, we demonstrated the outstanding performance of the hybrid capture method in detecting intra-host variants. We identified 229 intra-host variants at 182 sites in 18 fecal samples. Among them, nineteen variants presented frequency changes > 0.3 within 1–5 days, reflecting highly dynamic intra-host viral populations. Moreover, the evolution of the viral genome demonstrated that the virus was probably viable in the gastrointestinal tract during the convalescent period. Meanwhile, we also found that the same mutation showed a distinct pattern of frequency changes in different individuals, indicating a strong random drift. In summary, dramatic changes of the SARS-CoV-2 genome were detected in fecal samples during the convalescent period; whether the viral load in feces is sufficient to establish an infection warranted further investigation.

Molecular Diagnosis of Craniosynostosis Using Targeted Next-Generation Sequencing.

Genetic factors play an important role in the pathogenesis of craniosynostosis (CRS). However, the molecular diagnosis of CRS in clinical practice is limited because of its heterogeneous etiology. To investigate the genomic landscape of CRS in a Korean cohort and also to establish a practical diagnostic workflow by applying targeted panel sequencing. We designed a customized panel covering 34 CRS-related genes using in-solution hybrid capture method. We enrolled 110 unrelated Korean patients with CRS, including 40 syndromic and 70 nonsyndromic cases. A diagnostic pipeline was established by combining in-depth clinical reviews and multiple bioinformatics tools for analyzing single-nucleotide variants (SNV)s and copy number variants (CNV)s. The diagnostic yield of the targeted panel was 30.0% (33/110). Twenty-five patients (22.7%) had causal genetic variations resulting from SNVs or indels in 9 target genes (TWIST1, FGFR3, TCF12, ERF, FGFR2, ALPL, EFNB1, FBN1, and SKI, in order of frequency). CNV analysis identified 8 (7.3%) additional patients with chromosomal abnormalities involving 1p32.3p31.3, 7p21.1, 10q26, 15q21.3, 16p11.2, and 17p13.3 regions; these cases mostly presented with syndromic clinical features. The present study shows the wide genomic landscape of CRS, revealing various genetic factors for CRS pathogenesis. In addition, the results demonstrate that an efficient diagnostic workup using target panel sequencing provides great clinical utility in the molecular diagnosis of CRS.

Mo1741 PREVALENCE OF HUMAN PAPILLOMAVIRUS (HPV) INFECTION WITH HYBRID CAPTURE SCRENNING IN PATIENTS UNDERGOING ANUSCOPY AND FLEXIBLE RECTOSIGMOIDOSCOPY

HPV infection is currently one of the most prevalent sexually transmitted disease in the world. In Brazil, according to data from the Ministry of Health, it corresponds to 23.4% of these STD. Considered a pre-neoplastic condition, it has been a focus of attention in health policies because most patients are asymptomatic carriers. HPV detection can be performed by anuscopy associated with anal colposcopy or by evaluating the material obtained from the anal brush by the hybrid capture method. Evaluate the rate of HPV infection with hybrid capture in asymptomatic patients undergoing anuscopy and flexible rectosigmoidoscopy. Prospective study involving 252 patients without related symptons to HPV, with no suspicion at the time of the exam or prior history of HPV. The patients underwent HPV screening using hybrid capture in microplate to HPV from Group A-low-risk (6,11,42,43,44) and high-risk group B (16,18,31,33,3539,45,51,52,58,59,68) with the ability to detect 0.1 virus / cell copy during the examination. Patients with a previous history or diagnosis of HPV were excluded. From the 252 patients evaluated, 136 (53.96%) were male and 116 female, aged 16 to 71 years (mean: 37.4 years). HPV was detected in 50 patients (19.84%): HPV-A in 26 (10.32%) and HPV-B in 24 (9.52%). Of these (48%) presented pathologies at anuscopy, the most frequent being anal microfissures and hemorrhoidal disease. There was no significant difference in HPV prevalence in different age groups. Endoscopic exams may be an opportunity for HPV research in the anal region through this hybrid capture method. In a prospective study, our sample showed a prevalence of asymptomatic HPV infection in the anal region of 19.84%, of which 9.52% presented high-risk strains for cancer (HPV-B).

Genome-Wide Technologies to Study RNA-Chromatin Interactions.

An increasing number of studies have revealed that long non-coding RNAs (lncRNAs) play important roles in gene regulation and nuclear organization. Although the mechanisms are still largely unknown, many lncRNAs have been shown to interact with chromatin. Thus, one approach to understanding the function of these lncRNAs is to identify their sites of genomic interaction. Hybridization capture methods using oligonucleotide probes have been used for years to study chromatin-associated RNA. Recently, several groups have developed novel methods based on proximity ligation to investigate RNA–chromatin interactions at a genome-wide scale. This review discusses these technologies and highlights their advantages and disadvantages for the consideration of potential users.

Study on the correlation between hTREC and HPV load and cervical CINI/II/III lesions and cervical cancer.

ObjectiveTo investigate the correlation between hTREC and human papillomavirus (HPV) load and cervical intraepithelial neoplasia (CIN) grade II/III lesions and cervical cancer.MethodsA total of 135 patients with cervical lesions of different degrees admitted to our hospital from January 2016 to February 2017 were selected, including CIN I/III 65 cases, grade III 39 cases, and cervical cancer 31 cases. The expression of hTERC gene was detected by fluorescence in situ hybridization (FISH) in three groups, and the HPV load was detected by second‐generation hybridization capture (HC II) method, and its relationship with cervical lesion grade was analyzed. Department.ResultsThe positive expression rate of hTERC gene amplification was cervical cancer > CIN I/II lesion > CIN III lesion; the positive expression rate of HPV was cervical cancer > CIN I/II lesion > CIN III lesion. After treatment, the positive rate of hTERC gene amplification and HPV expression decreased significantly within 1 year (P < .05). Spearman's analysis showed that the degree of cervical lesion was positively correlated with hTREC and HPV load (P < .05).ConclusionhTREC and HPV are closely related to the occurrence and development of cervical precancerous lesions and cervical cancer. The abnormal amplification of hTERC gene increases with the grade of cervical lesions. Both of them can be used as auxiliary indicators for early screening, treatment, and prognosis of cervical cancer.

Implications of driver genes associated with a high tumor mutation burden identified using next‑generation sequencing on immunotherapy in hepatocellular carcinoma

Immune checkpoint blockade (ICB) therapy is a treatment strategy for hepatocellular carcinoma (HCC); however, its clinical efficacy is limited to a select subset of patients. Next-generation sequencing has identified the value of tumor mutation burden (TMB) as a predictor for ICB efficacy in multiple types of tumor, including HCC. Specific driver gene mutations may be indicative of a high TMB (TMB-H) and analysis of such mutations may provide novel insights into the underlying mechanisms of TMB-H and potential therapeutic strategies. In the present study, a hybridization-capture method was used to target 1.45 Mb of the genomic sequence (coding sequence, 1 Mb), analyzing the somatic mutation landscape of 81 HCC tumor samples. Mutations in five genes were significantly associated with TMB-H, including mutations in tumor protein 53 (TP53), Catenin®1 (CTNNB1), AT-rich interactive domain-containing protein 1A (ARID1A), myeloid/lymphoid or mixed-lineage leukemia (MLL) and nuclear receptor co-repressor 1 (NCOR1). Further analysis using The Cancer Genome Atlas Liver Hepatocellular Carcinoma database showed that TP53, CTNNB1 and MLL mutations were positively correlated with TMB-H. Meanwhile, mutations in ARID1A, TP53 and MLL were associated with poor overall survival of patients with HCC. Overall, TMB-H and associated driver gene mutations may have potential as predictive biomarkers of ICB therapy efficacy for treatment of patients with HCC.

Prognostic and predictive value of circulating tumour DNA (ctDNA) by amplicon-based next generation sequencing (NGS) of advanced pancreatic cancer (APC) in a phase I trial of oxaliplatin capecitabine and irinotecan (OXIRI) triplet chemotherapy.

730 Background: Tumoral KRAS mutations (KRAS mt) are detected in ~80% of APC and associated with a negative prognosis. Digital droplet PCR (ddPCR) has high sensitivity for circulating KRASmtbut narrower gene coverage. NGS using hybrid-capture methods has reported ctDNA KRASmtin ~30% of patients (pt). We previously presented clinical results of the Phase I trial of OXIRI (GI ASCO 18 #411). We now present the first prospective evaluation of ctDNA in APC by an amplicon-based NGS approach in this Phase I trial. Methods: Paired ctDNA and CA19-9 samples were taken at baseline, C2D1, C3D1 and end of trial. A targeted panel with error-correction (Lucence Diagnostics) was used to detect for mtin KRAS, TP53, SMAD4, CDKN2A, CTNNB1, GNAS, APC and MYC. CT scans were performed every 2 cycles. Survival curves by Kaplan Meier were compared by mutational status, ctDNA and CA19-9 response using the log-rank test. Spearman correlation of ctDNA and CA19-9 changes was performed. Results: ctDNA mtwas detected at baseline in 19/23 (83%) samples, comprising KRAS 17/23 (73%), TP53 (61%), SMAD4 (48%) and CDKN2A (30%). KRAS mtand SMAD4 mt conferred a negative prognosis for overall survival with a hazard ratio of 4.2 (CI: 1.6-10.4; p = 0.01) and 2.8 (CI: 0.9-8.65; p = 0.01) respectively. Drop in ctDNA and CA19-9 was associated with a trend for longer progression free survival at C2D1 (both) and C3D1 (ctDNA only). Radiological partial response (PR) was associated with lower ctDNA in 5/5 pt and CA 19-9 in 4/5 pt. Decrease in ctDNA and CA19-9 was associated with disease control (PR/SD) at C2D1 in 11/14 pt and 10/10 pt; at C3D1 in 11/12 pt and 6/7 respectively. No significant correlation between the amplitude of CA19-9 and ctDNA changes was found. Conclusions: ctDNA could be detected in 83% of pts of whom KRAS mtrates were similar to reports using tissue NGS. Determination of RAS mtand SMAD4 mtin ctDNA may aid in the prognostication of pts and decrease in ctDNA levels may predict for treatment benefit, similar in extent to CA 19-9. This may be particularly useful in non-CA19-9 secreting APC as an adjunct to imaging. Clinical trial information: NCT02368860.

Current Perspectives in Human Papilloma Virus: Where We are and What We Need?

It is now well-established that Human Papilloma Viruses (HPV) are responsible for causing &gt;90% of cervical cancers. Existing evidences have also demonstrated a key role for HPV in a portion of Head and Neck cancers as well as carcinomas of Vulva, Vagina, Penis and Anus. Therefore studies aiming at developing highly sensitive diagnostic methods have become high-priority in the recent years. To date, an estimated 202 types of HPV have been identified, of which only a small percentage viruses are involved in carcinogenesis. Among the carcinogenic HPV, the most predominant ones are HPV 16 and HPV 18. The other high risk types are 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70. In general, HPV is detected by polymerase chain reaction (PCR) and hybrid capture (HC) methods. However, the existing PCR methods specifically identify only HPV 16 and HPV 18, but not the other types. More over, the PCR protocols are known for errors, and are not suitable for on-field screening procedures, hence, a suitable much quicker and sensitive method is required at the earliest. More over, the PCR and HC methods are expensive, hence, require more funds to conduct public health screening campaigns. Therefore, in this book chapter we have made an attempt to address the recent developments in HPV screening methods, and covered various advanced procedures available in the literature to identify HPV in patient specimens. For example, a section is dedicated to cover the “Biosensors”, which have been developed in detecting the HPV DNA in body fluids. These devices are much simpler compared to sequencing technologies and likely to be the next-generation detection devices for HPV

Gambaran Hasil HPV Test dengan PAP Test pada Pasien dengan Lesi Prakanker Serviks di RSUP Dr M Djamil Padang

Objective: To determine the description of the HPV Test results with the PAP Test in patients with cervical precancerous lesions at Dr. M. Djamil Padang.Methods: This study used a descriptive method with a sample of all patients with cervical precancerous lesions who had the HPV test and PAP test results in Dr. M. Djamil Padang from January 1, 2015 - December 31, 2017. Data were taken from the patient's medical records, including diagnosis, PAP test results using the Hybrid Capture (HC) method, and HPV test. Results: During the study period, 80 patients had PAP test results leading to precancerous lesions and cervical cancer. Of the 80 patients, only 4 cases with positive HPV test results. Conclusion: In this study, there were many cervical precancerous lesions with negative HPV test results.Keywords: Cervical precancerous lesions, PAP test, the HPV test

Gambaran Hasil HPV Test dengan PAP Test pada Pasien dengan Lesi Prakanker Serviks di RSUP Dr M Djamil Padang

Objective : To determine the description of the HPV Test results with the PAP Test in patients with cervical precancerous lesions at Dr. M. Djamil Padang. Methods : This study used a descriptive method with a sample of all patients with cervical precancerous lesions who had the HPV test and PAP test results in Dr. M. Djamil Padang from January 1, 2015 - December 31, 2017. Data were taken from the patient's medical records, including diagnosis, PAP test results using the Hybrid Capture (HC) method, and HPV test. Results : During the study period, 80 patients had PAP test results leading to precancerous lesions and cervical cancer. Of the 80 patients, only 4 cases with positive HPV test results. Conclusion: In this study, there were many cervical precancerous lesions with negative HPV test results. Keywords: Cervical precancerous lesions, PAP test, the HPV test

Coleta de amostra para identificação de DNA do papilomavírus: conhecimento e habilidades

Resumo Objetivo Estabelecer a relação entre o conhecimento e as habilidades dos profissionais da saúde em coletar amostras para a identificação do DNA do papilomavírus. Métodos Estudo transversal, realizado entre março e junho de 2017, com uma população de 313 pessoas. A amostra probabilística foi calculada considerando um r=0,306, 95% de confiança e poder de 90%, equivalente a 90 trabalhadores de 23 centros de saúde. Um teste de conhecimento e um checklist foram administrados para avaliar as habilidades na coleta de amostra utilizando o teste de Captura Híbrida 2 (teste HPV-CH2). Para análise dos dados, foi utilizada a correlação de Pearson e ANOVA. Resultados Foi encontrada uma correlação entre conhecimento e habilidades (r=0,438) (p&lt;0,001). Quanto ao conhecimento, não houve diferença significativa nas médias obtidas nos três grupos de participantes (enfermeiros versus estagiários 3,27 p=0,557, médicos vs. enfermeiros 2,17 p=0,852, e médicos vs. estagiários 5,45 p=0,441). Quanto às habilidades, não houve diferença entre as médias obtidas entre médicos e enfermeiros (4,27 p=0,388), nem entre enfermeiros e estagiários (4,54 p=0,179), mas houve diferença entre médicos e estagiários (8,81 p=0,041). Conclusão Houve correlação entre o conhecimento e as habilidades em coletar amostras para a identificação do DNA do papilomavírus. Não houve diferenças significativas no conhecimento entre os grupos estudados, mas uma diferença em relação às habilidades foi encontrada entre médicos e estagiários.

CapTCR-seq: hybrid capture for T-cell receptor repertoire profiling

AbstractMature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis. We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing; clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the β and γ loci, respectively, whereas β locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays (r2 = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time.

An Enhanced Sensitivity DNA Sequencing Protocol for the Detection in AML of Measurable Residual Disease (MRD) Applicable for All Mutations

Background: One of the more important prognostic factors used to predict the outcome in acute myeloid leukemia (AML) is the persistence of leukemic cells after treatment. The reliable measurement of residual disease (MRD) offers many other clinical uses besides. An assay that was facile, affordable, and applicable to the broadest group of patients would find immediate favor.Next-generation sequencing (NGS) combined with various enrichment methods allow high sequencing depth on restricted targets. For instance, by enriching for <100 targets raw sequencing depths of 105 to 106 are routinely achieved. However, after excluding PCR duplicates identified using either end-point diversity of paired-end fragments or molecular tags, we found that NGS libraries effectively capture only a few percent of the input genomes (typically 1000 genomes or fewer for a 100ng input DNA corresponding to 30,000 genomes) effectively restricting the limit of detection (LOD) to approximately 1%. Hybrid capture and primer extension methods including protocols available from Nimblegen, Illumina for whole exome sequencing, exon capture using IDT probes and Nugen yielded similar results: all showed extensive loss of input genome diversity restricting the LOD (see Table 1 Section A). Most surprising was whole exome sequencing (WES). Sequencing 1000 ng input representing 300,000 genomes performed worst: for a given locus, loss of genomic diversity of approximately can be as high as 99.99% and is always more than 95% (Table 1A). More input is clearly not better.Methods and Results: We used the ThermoFisher multiplex PCR-based method to capture the 50 genomic fragments most frequently mutated in AML and labeled each with unique DNA tags. Sequence generated on an Ion Torrent showed that for 100ng of input DNA, >50% of the genomic diversity of that input DNA was preserved, i.e., >15,000 unique genomes were captured and sequenced from an initial 30,000 genomes resulting in a LOD of 0.1% with high precision as a mutation present at 0.1% in 15,000 genomes could be observed ~15 times. As shown in Table 1B, this method scales (non-linearly) as input DNA is increased; samples containing 500ng of DNA (~150,000 genomes) reproducibly permit an LOD of 0.01%, a sensitivity far exceeding any other non-allele specific method for measuring residual disease.In a real world application, we followed 3 AML patients from diagnosis to clinical CR. We tracked all mutations present at diagnosis using both IDT hybrid capture (HC) and the ThermoFisher (TF) method. One patient had molecular evidence of residual disease (VAF3%) that was detected and similarly quantitated by both HC and TF, in the second patient mutation were present at 1% by HC and 3% by TF . The TF value is the reliable one as it is derived from multiple independent templates. Finally the third patient was in molecular remission by HC but had a mutation allele detected by the TF with a frequency of 0.07%. We have extended these studies to include more than a dozen patients followed through relapse with similar results.These pilot studies provide clear evidence that standard sequencing methods cannot reliably promise LODs below 1%; further, the ThermoFisher method enhances the LOD at least 20-fold in an assay that can be applied to the great majority of AML patients. DisclosuresRienhoff:Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Natsoulis:Imago BioSciences, Inc.: Consultancy, Equity Ownership. Jones:Imago BioSciences, Inc.: Employment, Equity Ownership. Peppe:Imago BioSciences, Inc.: Employment, Equity Ownership. Cao:Thermo Fisher Scientific: Employment. Hanif:Thermo Fisher Scientific: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding.

An Improved NGS Library Construction Approach Using DNA Isolated from Human Cancer Formalin-Fixed Paraffin-Embedded Samples.

Identification of genomic alterations from formalin-fixed paraffin-embedded (FFPE) samples using next-generation sequencing (NGS) is very important for cancer-targeted therapy today. To achieve a higher efficiency and shorter turn-around time for NGS library preparation, here, we compared NGS library preparation processes and outcomes with three commercial library construction methods and two hybridization capture methods thus, developed an improved NGS library construction approach. This improved approach took advantage of both methods and resulted in a higher output from the same input DNA, including higher library construction success rate, higher probe capture rate, and shorter turn-around time. Using this approach, targeted region libraries could be constructed within only 1 day for FFPE samples; therefore, this approach has potential applications of NGS in routine clinical tests. Anat Rec, 302:941-946, 2019. © 2018 Wiley Periodicals, Inc.

Abstract 1626: Sensitive detection of MET exon 14 skipping by RT-qPCR and next generation sequencing

Abstract The MET receptor tyrosine kinase (MET) gene is a proto-oncogene whose abnormal activation can trigger tumor growth, angiogenesis, and metastasis. Numerous mutations have been identified that lead to over-activation of the gene, including well characterized amplification and somatic mutations. Mutations that lead to the skipping of MET exon 14 are likely to be highly predictive of response to MET inhibition. Exon 14 contains a motif that is required for the efficient recruitment of the ubiquitin ligase, CBL, which targets MET for degradation. Loss of exon 14 leads to prolonged stability of the MET protein which causes increased signaling upon stimulation by hepatocyte growth factor. Over 200 distinct mutations have been identified that cause MET exon 14 skipping ranging from single nucleotide mutations to large insertion/deletions. Due to this complexity, detecting MET exon 14 skipping mutations using DNA-based NGS hybridization capture methods is challenging. RNA-based testing can circumvent the complexity of DNA-based changes. We developed a RT-qPCR assay that detects MET exon 14 skipping independent of the causal mutation. Analytical validation using RNA admixtures demonstrated 1% sensitivity of detecting MET exon 14 mutant transcripts. 100 FFPE lung adenocarcinomas were screened with the RT-qPCR assay. Of 100, two samples (2%) were identified as positive for MET exon 14 skipping. The two positive samples along with three negative samples were orthogonally confirmed using the Illumina TruSight Tumor 170 hybridization capture-based targeted panel assay that simultaneously analyzes DNA and RNA. Overall, the number of samples identified positive for MET exon 14 by RT-qPCR test is in line with the frequency observed from other studies in lung adenocarcinomas of about ~3%. The assay can detect as little as 1% transcripts with MET exon 14 skipping suggesting that we have developed an accurate and sensitive RNA-based assay for use in clinical trials. Citation Format: Mukund Patel, Honey Polur, Patrick Hurban. Sensitive detection of MET exon 14 skipping by RT-qPCR and next generation sequencing [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 1626.

Massive Parallel Regression: A Précis of Genetic Mechanisms for Vision Loss in Diving Beetles.

Two tribes of subterranean dytiscid diving beetles independently colonized groundwater systems of the Western Australian arid zone, a habitat transition that was most likely driven by the contraction of surface water bodies following late Neogene aridification of the Australian continent. These "stygofauna" are now trapped within discrete calcrete aquifers that have formed in paleodrainage valleys, resulting in the world's most diverse radiations of subterranean dytiscid beetles. Approximately 100 species from three genera exhibit partial or fully regressed visual systems and are essentially blind. This unique study system, with multiple independent transitions to subterranean life enables regressive and adaptive evolutionary processes to be studied in parallel at an unheralded comparative scale. Here we provide an overview of the progression of dytiscid beetle research and undertake a literature survey of published research within the field of regressive evolution as it applies to eye loss. We detail our exploration of insect vision genes for signatures of adaptive and neutral evolutionary mechanisms related to eye regression, largely within photoreceptor and eye pigment genes. Our project makes use of transcriptome data from five representative dytiscid beetle species (two surface and three subterranean) in order to design a customized set of RNA baits for use in a hybrid-capture method to target a pool of vision genes sequenced using high-throughput Illumina platforms. This methodological design permits the assessment of modifications in the genomic sequence of beetle vision genes at a much broader scale than Sanger sequencing, enabling a higher number of both target species and genes to be simultaneously assessed relative to research time-investments. Based on our literature search criteria of the research field ("regressive evolution" + "eyes"), 81 papers have been published since the late 1980s accruing an h-index of 27 and a mean citation rate of 24.57. Collective annual citations for this field of research have surged over the past 5 years, an indication that broader scientific community interest is gaining momentum. The majority of publications (75%) have focused on the chordate clade Actinopterygii. Historically, research on variant subterranean taxa has faced difficulties inferring the evolutionary mechanisms of eye regression (and vision loss) using molecular approaches because only a handful of target genes could be feasibly addressed within grant funding cycles. From a comparative phylogenetic perspective, next-generation sequencing approaches applied to stygobiontic dytiscid beetles hold the potential to greatly improve our understanding of the genetic mechanisms underlying regressive evolution generally.

An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin‐fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter‐laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next‐generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.

Catching RNAs on chromatin using hybridization capture methods.

The growing appreciation of the importance of long noncoding RNAs (lncRNAs), together with the awareness that some of these RNAs are associated with chromatin, has inspired the development of methods to detect their sites of interaction on a genome-wide scale at high resolution. Hybridization capture methods combine antisense oligonucleotide hybridization with enrichment of RNA from cross-linked chromatin extracts. These techniques have provided insight into lncRNA localization and the interactions of lncRNAs with protein to better understand biological roles of lncRNAs. Here, we review the core principles of hybridization capture methods, focusing on the three most commonly used protocols: capture hybridization analysis of RNA targets (CHART), chromatin isolation by RNA purification (ChIRP) and RNA affinity purification (RAP). We highlight the general principles of these techniques and discuss how differences in experimental procedures present distinct challenges to help researchers using these protocols or, more generally, interpreting the results of hybridization capture experiments.

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

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