Next-generation Sequencing Libraries Research Articles

Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis.

Single next-generation sequencing (NGS) proved to be an important tool for monitoring the SARS-CoV-2 outbreak at the global level Until today, thousands of SARS-CoV-2 genome sequences have been published at GISAID (Global Initiative on Sharing All Influenza Data) but only a portion are suitable for reliable variant analysis. Here we report on the comparison of three commercially available NGS library preparation kits. We discuss advantages and limitations from the perspective of required input sample quality and data quality for advanced SARS-CoV-2 genome analysis.

Abstract 1988: Enabling personalized biomarker discovery in challenging oncology samples by coupling a novel library preparation chemistry with hybridization capture

Abstract Discovery and identification of new, targetable biomarkers is driven by comprehensive tumor profiling using next generation sequencing (NGS). However, converting tissue samples into NGS libraries is often challenging due to the low quantity and quality of DNA in such samples. Here we present sensitive and accurate detection of low-frequency variants by combining a novel library preparation optimized for low-input and degraded samples with IDT's xGen hybridization capture. The library preparation is enabled by an engineered mutant ligase and proprietary sequencing adapters that together prevent chimeras, suppress dimer-formation, enable double strand consensus calling, and maximize conversion. We demonstrate the performance of these reagents with archived tissue samples collected from five individuals with lung cancer. These matched samples consisted of DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor, DNA isolated from fresh-frozen normal tissue, and cell-free DNA (cfDNA) isolated from plasma. Sequencing libraries were prepared from 250 ng of tumor and healthy samples and 10 ng of cfDNA. Libraries were captured using a pan-cancer panel designed to simultaneously detect copy number variations, indels, rearrangements, and microsatellite instability across 532 oncogene targets. For each individual, tumor-associated mutations were identified using single-stranded consensus calling of variants found in tumor but not normal samples. As cfDNA has been correlated to disease progression, we wanted to determine if tumor-associated variants could be identified in the matched cfDNA samples. Custom capture panels were designed and delivered within 5 business days to target each individual's tumor-associated mutations. Relevant cfDNA samples were captured with these custom panels, sequenced, and variants were called after double stranded consensus calling. Mutations called from both FFPE tumor and cfDNA samples were verified by droplet digital PCR. By combining a high conversion library preparation with efficient hybridization capture and double stranded consensus calling, we have demonstrated an effective approach to extract information from difficult samples. Citation Format: Karissa Scott, Ushati Das Chakravarty, Hsiao-Yun Huang, Timothy Barnes, Kevin Lai, Jessica Sheu, Tzu-Chun Chen, Ramses Lopez, Lynette Lewis, Anastasia Potts, Steven Henck. Enabling personalized biomarker discovery in challenging oncology samples by coupling a novel library preparation chemistry with hybridization capture [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1988.

Abstract 1352: Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers

Abstract Hybrid capture is a popular and well-accepted method to select targeted genomic regions for subsequent next generation sequencing (NGS). Despite its popularity in research and clinical diagnostics, hybrid capture NGS is now facing the challenge by the high throughput demands generated by Novaseq, Illumina's flagship platform that occupies the research and industry market rapidly in recent years. Novaseq create tetrabytes of sequencing data per run, allowing unprecedented level of sample multiplexing, especially for targeted NGS. Conventional blockers, whose sample index-matching region uses either degenerate bases or universal pairing bases, still need to match the length of the sample index for each individual library. When using NovaSeq, scientists need to combine huge number of samples using different types of index system in each single run. However, matching different hybridization blockers with corresponding index system is time consuming and error-prone. Here we introduce a novel design and chemistry to hybrid capture blockers to maximized the pooling capacity of hybrid capture to match the increasing need of sample throughput and multiplexing. Our new hybrid capture blockers are not only an essential component in the target enrichment workflow to block the permissive interactions between library adapters and target probes, which may lower the capturing efficiency and generate excessive data from the off-target sequence, but also significantly promotes the capturing capacity and efficiency, and simplifies the workflow. Our new blocker, GenFisher Universal Blockers, using our proprietary design and chemistry, create a novel solution to the conjugation between sample indexes on libraries and blockers, enabling blockers bind adapters regardless to the index length and sequence. In our experiment, NGS libraries are created with a commercially available kit and TruSeq style adapters that contain sample indexes with 3 different lengths: 6nt, 8nt and 10nt. Hybrid captures were then carried out with 8,000-probe GenFisher hybridization panel and kit and Universal Blockers for each individual libraries and the combined library with different indexes. Sequencing data showed that the on-target base percentage was consistently at <75% for all captures for both individual or combined multiplex libraries, whereas hybrid capture without blockers yielded >30% on-target rate. We also compared GenFisher Universal Blockers with the blockers from two other industrial leading providers, which are also claimed to be ‘universal'. Our data showed that GenFisher Universal Blockers was the only truly universal solution that worked with all 3 index lengths unbiasedly while the other two are still index length-sensitive and struggled with 10nt indexes library capturing (on-target ~50%, compared to <65% at 6/8nt). With its versatility, GenFisher Universal Blockers enables higher multiplexing capacity, as well as the freedom of index selection to hybrid capture NGS users. Citation Format: Jianpeng Wang, Long Fan, Lumeng Ye, Jiashi Wang, Irene Song, Cedric Wu. Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1352.

Abstract 3619: High quality library preparation and accurate sequencing from highly damaged matched patient sample sets

Abstract Preparation of high quality next-generation sequencing (NGS) libraries is critical for accurate identification of disease-causing mutations, but clinical scientists are often limited by the input DNA quality. In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy, and in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and extensive damage from fixation, storage, and extraction methods. Sequencing artifacts arising from damaged DNA bases raise the level of apparent mutations, making it more difficult to discern true, low frequency, disease-related variants from noise. We developed a second-generation, multi-enzyme DNA repair mix (V2) that is designed to repair the most prevalent damage types found in FFPE DNA including cytosine deamination, oxidative damage, abasic sites, nicks, and gaps. We tested the application of the V2 enzymatic repair mix in Illumina library preparation for DNA samples extracted from a series of matched patient sample sets, which included: FFPE normal and tumor tissue, and frozen normal and tumor tissue for each patient. Following this, we performed target enrichment, deep sequencing, and variant calling analyses. Our library preparation workflow integrates enzymatic repair upstream of Illumina library preparation without an additional bead purification step, maximizing library yield and complexity from precious FFPE samples. Rescue of damaged DNA molecules for library conversion and repair of damaged DNA bases improved on-target rate, coverage uniformity, and duplication rates. With a matched frozen sample for each FFPE specimen, we demonstrate that repair of damaged DNA bases improves sequencing accuracy by reducing the number of false-positive variant calls. We propose that enzymatic repair enables researchers to capture as much useable, accurate data from poor quality FFPE samples as possible. Citation Format: Margaret R. Heider. High quality library preparation and accurate sequencing from highly damaged matched patient sample sets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3619.

Abstract 2293: A urine-based liquid biopsy method for detection of upper tract urinary carcinoma

Abstract Background: Upper tract urinary carcinoma (UTUC) includes renal pelvic cancer and ureteral cancer. For its diagnosis, ureteroscopy is the only method to obtain pathological result before surgery. However, there are also some problems with ureteroscopy. In addition to the trauma and infection risks, the possibility of exudation and adhesion of surrounding tissues after ureteroscopy is significantly increased, hypertension in the cavity may cause tumor spread, and the use of ureteroscopy and biopsy may cause recurrence of bladder tumors after surgery. These unfavorable factors make clinically urgent need for a sensitive and non-invasive method for the diagnosis of UTUC. Materials and Methods: In this study, 150 hematuria patients with upper tract urinary disease and 100 healthy people were enrolled. Among the patients, 75 were pathologically confirmed with upper tract urinary carcinoma while 75 with benign urinary diseases. A Genetron Urinary Assay was designed to maximize the number of unique driver gene variants of UTUC by a limited number of amplicons. Next-generation sequencing libraries were constructed using multiplex PCR methods. We also detected CpG-sites on ONECUT2 gene by performing bisulfite-specific real-time PCR for bisulfite converted DNA. Furthermore, we developed a prediction model based on several significant features to evaluate the risk of UTUC. Results: 234 samples were available for analysis. Our liquid biopsy assay demonstrated a sensitivity of 94%, a specificity of 96%, a positive predication value (PPV) of 92% and a negative predication value(NPV) of 98% in this study. By using a decision tree model, we found age, gene mutations of HRAS, TERT and TP53 and methylation of ONECUT2 did place at some key nodes to classify the risk of patients of UTUC, and age around 48.5, the frequencies of mutated genes above certain value and ΔCt value of ONECUT2 about 7.6 would be good cutoffs. This model was able to stratify patients into a low or high-risk group with an accuracy score at 94%. After further analysis, we found that ONECUT2 methylation and TP53 mutations were more common in high grade UTUC (p<0.01), while AKT1 and FGFR3 (trend, but no statistical significance) mutations were more often shown in low grade group. No significant difference of gene mutations frequency was found between non muscle invasive and muscle invasive group, but FGFR3 as well as TERT and PIK3CA gene showed a trend of higher occurrence. Conclusion: In this study, Genetron Urinary Assay has a good performance in the diagnostic of UTUC. Its high sensitivity, high specificity, and non-invasiveness make it a potential clinical alternative to ureteroscopy. Further validation in a large prospective cohort of wide population is necessary to prove the true clinical value of this newly developed method. Citation Format: Xin Ma, Yansheng Xu, Hongzhao Li, Xing Ai, Jiangping Gao, Yiming Liang, Qiaosong Zheng, Kaisheng Mao, Tonghui Ma, Xu Zhang. A urine-based liquid biopsy method for detection of upper tract urinary carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2293.

Abstract 5429: Next generation sequencing of clinical samples from more than 4200 patients with myeloid malignancies

Abstract Myeloid malignancies can be defined as a series of conditions associated with abnormal myeloid cell proliferation and/or differentiation. This group of disorders is heterogeneous and includes acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and others. These disorders are often driven by somatic mutations and the detection of genetic variants can have diagnostic, prognostic, and therapeutic relevance to the patient. A laboratory developed test (IntelliGEN Myeloid) based on a next generation sequencing (NGS) panel that targets mutations within 50 genes linked to myeloid malignancies was developed, validated, and clinically implemented. Within these genes, multiple variant classes including single nucleotide variants, insertions, and deletions were reported. Whole blood or bone marrow samples from more than 4200 patients were submitted for testing during the evaluation period. DNA was extracted from each sample and used as the template for creating targeted NGS libraries (ArcherDx, Boulder, CO) that were subsequently sequenced on Illumina DNA sequencers (Illumina, San Diego, CA). Results were reviewed, orthogonally confirmed unless previously validated, and reported by clinical laboratory directors. Herein, the anonymized results from this cohort are presented. While testing is ongoing and additional samples will be included in the final analysis, these data were censored to include 4270 patient samples with reported results at the time of submission. The median patient age in this cohort was 71 years with 56.9% being male. Actionable variants (reported as Tier-I/II) or variants of unknown significance (reported as Tier III) were identified in all 50 genes within the panel, consistent with a comprehensive panel being necessary to encompass the mutations contributing to the etiology of these diseases. In total, more than 4400 unique variants were observed in this sample cohort. Clinically or potentially relevant variants (Tier I, II, or III) were detected in the majority of patients tested (72.8%) with a mean of 3.3 variants reported per patient. The size of our cohort also enabled indication-specific evaluation of mutational profiles. In patients where the indication for testing included AML, the most commonly mutated genes were TET2, ASXL1, DNMT3A, and RUNX1 whereas that list included ASXL1, DNMT3A, SF3BP1, SRSF1, and TP53 in patients where MDS was listed as an indication for testing. Taken together, these data describe the clinical testing demographics, the mutational spectrum of myeloid malignancies in a large clinical cohort, and suggest that mid-size NGS panels may provide physicians with diagnostic, prognostic, and therapeutic information that can help facilitate patient care. Citation Format: Taylor J. Jensen, Heidi M. Hoffman, Angela Leo, Michael Mooney, Sabrina Gardner, Wenjie Chen, Narasimhan Nagan, Deborah Boles, Scott Parker, Tamara J. Richman, Stanley Letovsky, Henry Dong, Li Cai, Anjen Chenn, Marcia Eisenberg. Next generation sequencing of clinical samples from more than 4200 patients with myeloid malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5429.

Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA

Abstract There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications. Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5363.

Abstract 1364: Spatially-resolved in situ expression profiling using the GeoMx™ Cancer Transcriptome Atlas panel in FFPE tissue

Abstract The emerging field of spatial genomics represents a significant advance for biology. To drive new discoveries in spatial genomics and immuno-oncology, we introduce the GeoMx Cancer Transcriptome Atlas (CTA) Panel for comprehensive spatial analysis of cancer pathways using the Nanostring GeoMx Digital Spatial Profiler (DSP). We demonstrate profiling of 1600+ immuno-oncology targets in the tumor, microenvironment, and immune compartments of archival FFPE tissue sections, coupled to downstream Next Generation Sequencing (NGS) readout to enable high-throughput workflows. High-plex spatial RNA molecular profiling with GeoMx CTA was performed as follows: 1. Photocleavable DNA oligonucleotides tags were coupled to 8000+ in situ hybridization probes targeting 1600+ genes. These reagents were allowed to bind targets directly on slide-mounted FFPE tissue sections. 2. ROIs were identified and selected using GeoMx DSP, and ROI-specific oligonucleotide tags were released using ultraviolet exposure. 3. Released oligonucleotide tags from each ROI were collected and deposited into designated wells on a microtiter plate, allowing well indexing of each ROI during NGS library preparation. 4. After indexing, the entire plate was pooled into a single tube for purification and then sequenced on an Illumina instrument. 5. NGS reads were processed into digital counts and mapped back to each ROI, generating a map of transcript activity within the tissue architecture. We compared data from experiments in which bulk RNA-seq and GeoMx DSP using the CTA Panel were performed on the same samples. Overall, we found good correlation between pseudo-bulk GeoMx CTA (sum of ROIs) and RNA-seq from the same tissue specimen. Individually, however, each ROI showed a distinct expression pattern from bulk, and ROI expression patterns clustered based on similar tissue morphology. Importantly, GeoMx CTA was able to detect a higher number of genes with low expression within the microenvironment and immune spatial compartment compared to bulk RNA-seq, providing a detailed look at the anti-tumor immune response. Lastly, we profiled similar tissues using a novel 18000+ gene whole transcriptome panel and found further enrichment of low-expressers relative to RNA-seq, revealing novel spatial biology previously masked by bulk assays. Together, these data demonstrate that GeoMx offers high sensitivity for genome-scale expression profiling while preserving critical information about tissue architecture. GeoMx DSP technology is for Research Use Only and not for use in diagnostic procedures. Citation Format: Margaret L. Hoang, Michelle Kriner, Zoey Zhou, Zach Norgaard, Kristina Sorg, Chris Merritt, Erin Piazza, Marty Ross, Robin Fropf, Nileshi Saraf, Patrick Danaher, Michael Rhodes, Joseph Beechem. Spatially-resolved in situ expression profiling using the GeoMx™ Cancer Transcriptome Atlas panel in FFPE tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1364.

Abstract 730: Validation of a NGS-based MSI testing workflow in liquid biopsy

Abstract Background Microsatellite instability (MSI) is a molecular phenotype characterized by the lengthening or shortening of tandem DNA repeats within the human genome. MSI phenomenon has been reported in various solid tumors as the consequence of DNA mismatch repair deficiency. It is generally associated with good prognosis and effective immunotherapy treatment. The current MSI tests are based on various methodologies, including immunohistochemistry, polymerase chain reactions, or next-generation sequencing (NGS). Each testing methodology has its own technical advantages, but they either fail at liquid biopsy specimen or implement complicated and expensive experimental interventions. Methods A designated analysis pipeline was developed individually for tissue biopsy and liquid biopsy to evaluate the stability of MSI sites within a NGS panel testing targeting over 400 genes at a common depth of coverage (<1000X for tissue biopsy and <5000X for liquid biopsy). The liquid biopsy algorithm implements a signal enhancing module based on a pool of normal samples to boost assay performance. We validated the tissue biopsy testing with an exploratory cohort of 90 gastrointestinal tract cancer patients. Retrospective review of over 10,000 pan-cancer patient-matched tissue-cfDNA sample pairs permitted a comprehensive evaluation of the liquid biopsy MSI testing performance. Results For tissue biopsy specimen validation, the assay system displayed 0.97 area-under-the-curve (AUC) in receiver operative characteristics (ROC) curve analysis against the comparator assay, out-performing publicly available MSI analysis software. Unfortunately, the tissue MSI analysis pipeline and publicly available software fails with less than 10% sensitivity and 0.55 AUC when applied to liquid biopsy specimen with the patient-matched tissue MSI status used as the gold standard. In contrast, the dedicated cfDNA analysis pipeline significantly boosted the assay performance to 0.73 without additional experimental interventions, such as wild-type allele depletion or molecular barcode. Conclusions MSI testing in liquid biopsy specimen remains challenging due to the low fraction of tumor originated cfDNA and consequently weak signal intensity. The implementation of the signal enhancing MSI analysis algorithm demonstrated high proficiency at improving the signal-to-noise ratio. This bioinformatics approach offered a cost-effective and non-invasive alternative for MSI testing without significantly modifying the NGS library preparation protocols. Citation Format: Xiangyuan Ma, Hua Bao, Xiaoling Tong, Zhili Chang, Xunbiao Liu, Xiuyan Zhang, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Validation of a NGS-based MSI testing workflow in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 730.

Abstract 3099: Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients

Abstract Purpose Circulating tumor DNA (ctDNA) can potentially be used to monitor cancer treatment response. In this study, we developed and used a sensitive and selective ultra-deep next-generation sequencing (NGS) approach for detection of ctDNA in lung cancer patients. Method Most studies use fixed gene panels for detection of ctDNA by NGS. Deep sequencing of large target regions is expensive and generates a myriad of variants, which are difficult to interpret. For the purpose of using ctDNA for monitoring, we try to overcome these challenges by sequencing only selected regions where mutations were previously found in the tumor. This method is more sensitive and cost-efficient. In this study, we included about 80 lung cancer patients. Tumor DNA was sequenced with a large gene panel and somatic mutations were identified in each tumor. We then defined the target region according to the tumor mutations and selected primers for constructing NGS libraries. Since the coverage region is small it enables ultra-deep sequencing of several samples simultaneously. We evaluated the lower limit of detection for this method by sequencing a series of artificially constructed samples with known mutant allele frequencies (MAFs). DNA from peripheral blood leucocytes was sequenced in parallel with plasma DNA to exclude variants from clonal hematopoiesis. Preliminary results Using 40 ng as input DNA, which contains approximately 12,000 haploid genomes, we detected mutations down to 0.02% MAF. This corresponds to about 6 mutated copies per mL plasma. Currently, we are applying this method on patient plasma DNA samples. Conclusion We have developed a sensitive method for ctDNA detection, and we will present the results of the lung cancer patient samples. Citation Format: Anine Larsen Ottestad, Elisabeth F. Emdal, Sissel G. Wahl, Bjørn Henning Grønberg, Hong Yan Dai. Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3099.

A universal and independent synthetic DNA ladder for the quantitative measurement of genomic features

Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure diverse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.

A smart polymer for sequence-selective binding, pulldown, and release of DNA targets

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved the data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets.

Molecular characteristics and markers of advanced clear cell renal cell carcinoma: Pitfalls due to intratumoral heterogeneity and identification of genetic alterations associated with metastasis.

To identify clear cell renal cell carcinoma-related gene mutations potentially associated with aggressive disease, sarcomatoid differentiation or poor prognosis. We carried out genomic analysis of 217 tumor foci from 25 patients with conventional clear cell renal cell carcinoma (14 patients), clear cell renal cell carcinoma with sarcomatoid differentiation (six patients) and non-clear cell renal cell carcinoma (five patients). Each tumor nodule on the tissue block that corresponded to the same focus on the slide was separated from the normal parenchyma and other histologically distinct areas of tumor. The isolated tumor foci were used for subsequent analyses and sequencing. Deoxyribonucleic acid from the formalin-fixed paraffin-embedded tissues was extracted. Multiplex bar-coded polymerase chain reaction amplification was carried out using next-generation sequencing libraries. Overall, 67 protein alterations, including amino acid alterations, frame shifts and splice site mutations in seven genes were identified in the cohort of renal cell carcinoma tumors included in this study. Fewer patients with clear cell renal cell carcinoma with sarcomatoid differentiation had clear cell renal cell carcinoma-related mutations in comparison with patients with conventional clear cell renal cell carcinoma. Additionally, the average number of unique clear cell renal cell carcinoma-related protein alterations per patient was significantly lower in clear cell renal cell carcinoma with sarcomatoid differentiation than in conventional clear cell renal cell carcinoma. Mutations in PBRM1 were identified in a higher proportion of patients with high-grade tumors (World Health Organization/International Society of Urological Pathology grade4) and in the primary tumors of six of 10 (60%) patients with metastatic disease. Although there are pitfalls due to intratumoral heterogeneity and sampling bias, mutations in PBRM1 may be associated with metastasis and aggressive disease in clear cell renal cell carcinoma.

Identification of sulfate-reducing and methanogenic microbial taxa in anaerobic bioreactors from industrial wastewater treatment plants using next-generation sequencing and gene clone library analyses

An understanding of microbial communities present in anaerobic bioreactors can strongly facilitate the development of approaches to control undesirable microorganisms, such as sulfate-reducing bacteria (SRB), in the system. In this study, overall microbial communities present in anaerobic bioreactors from seven industrial wastewater treatment plants (including food, pulp and paper industries) were investigated using 16S rRNA gene amplicon sequencing (MiSeq, Illumina). The dominant methanogens identified in the anaerobic bioreactors treating industrial wastewater were Methanobacterium and Methanosaeta; Methanospirillum was a predominant methanogen in the anaerobic sludge digester. Hydrogenotrophic and acetoclastic methanogens were detected at similar relative abundances in the anaerobic covered lagoons treating starch wastewater, whereas hydrogenotrophic methanogens were the predominant methanogens present in the sludge digester. SRB communities were further investigated using dsrB gene clone libraries. The results indicated the presence of SRB, such as uncultured Desulfobulbus sp., Syntrophobacter fumaroxidans, Syntrophorhabdus sp. PtaB.Bin027, and Desulfovibrio fructosivarans JJ. Incomplete-oxidizing SRB were the predominant SRB in all of the anaerobic bioreactors treating wastewater. In contrast, similar relative abundances of complete and incomplete-oxidizing SRB were observed in the sludge digester. The results of this study can further facilitate the development of SRB-controlling strategies to improve the efficiency of wastewater treatment.

Novel NGS pipeline for virus discovery from a wide spectrum of hosts and sample types

The study of the microbiome data holds great potential for elucidating the biological and metabolic functioning of living organisms and their role in the environment. Metagenomic analyses have shown that humans, along with for example, domestic animals, wildlife and arthropods, are colonized by an immense community of viruses. The current Coronavirus pandemic (COVID-19) heightens the need to rapidly detect previously unknown viruses in an unbiased way. The increasing availability of metagenomic data in this era of next-generation sequencing (NGS), along with increasingly affordable sequencing technologies, highlight the need for reliable and comprehensive methods to manage such data. In this article, we present a novel bioinformatics pipeline called LAZYPIPE for identifying both previously known and novel viruses in host associated or environmental samples and give examples of virus discovery based on it. LAZYPIPE is a Unix-based pipeline for automated assembling and taxonomic profiling of NGS libraries implemented as a collection of C++, Perl, and R scripts.

Influence of long-term organic and conventional fertilization on bacterial communities involved in bioelectricity production from paddy field-microbial fuel cells.

Soils are rich in organics, particularly those that support the growth of plants. These organics are possible sources of sustainable energy, and a microbial fuel cell (MFC) system can potentially be used for this purpose. In this, the soil organic content expelled from plant root was possibly converted into electrical energy through the microbial metabolic process. The integration of MFC systems with living plant root system is a novel approach, which will facilitate sustainable resource for energy production. Therefore, the objective of this study is to electrochemically evaluate the paddy field MFCs (PF-MFCs) performance and methane emission under organic and conventional fertilization systems in paddy fields, and its impact on bacterial communities involved in bioelectricity production. Graphite (anode) and carbon (cathode) electrode MFC systems were configured and assembled in organic and conventionally fertilized paddy fields. The anode and bulk soil-associated bacterial communities were examined using high-throughput Illumina MiSeq sequencing platform. Our results revealed that the maximum electricity production and power density were observed in CFPF-MFC with less methane emission compared to OFPF-MFC. The next-generation sequencing (NGS) libraries showed that the bacterial population was significantly increased in the organic-fertilized field and the enhanced occurrence of the Geobacteraceae family in CFPF-MFC anode. By enhancing Geobacteraceae occurrence on the anode, the conventional fertilization improved the bioelectricity production with less methane emission. Further extension in the establishment of plant MFCs in various sedimentary environments will solve the global energy crisis.

Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome.

Long interspersed element-1 retrotransposons (LINE-1 or L1) are ∼6 kb mobile DNA elements implicated in the origins of many Mendelian and complex diseases. The actively retrotransposing L1s are mostly limited to the L1 human specific (L1Hs) transcriptional active (Ta) subfamily. In this manuscript, we present REBELseq as a method for the construction of Ta subfamily L1Hs-enriched next-generation sequencing libraries and bioinformatic identification. REBELseq was performed on DNA isolated from NeuN+ neuronal nuclei from postmortem brain samples of 177 individuals and empirically-driven bioinformatic and experimental cutoffs were established. Putative L1Hs insertions passing bioinformatics cutoffs were experimentally validated. REBELseq reliably identified both known and novel Ta subfamily L1Hs insertions distributed throughout the genome. Differences in the proportion of individuals possessing a given reference or non-reference retrotransposon insertion were identified. We conclude that REBELseq is an unbiased, whole genome approach to the amplification and detection of Ta subfamily L1Hs retrotransposons.

Parallel DNA Extraction From Whole Blood for Rapid Sample Generation in Genetic Epidemiological Studies.

Large-scale genetic epidemiological studies require high-quality analysis of samples such as blood or saliva from multiple patients, which is challenging at the point of care. To expand these studies’ impact, minimal sample storage time and less complex extraction of a substantial quantity and good purity of DNA or RNA for downstream applications are necessary. Here, a simple microfluidics-based system that performs genomic DNA (gDNA) extraction from whole blood was developed. In this system, a mixture of blood lysate, paramagnetic beads, and binding buffer are first placed into the input well. Then, the gDNA-bound paramagnetic beads are pulled using a magnet through a central channel containing a wash buffer to the output well, which contains elution buffer. The gDNA is eluted at 55°C off the chip. The 40-minute microfluidic protocol extracts gDNA from six samples simultaneously and requires an input of 4 μL of diluted blood and a total reagent volume of 75 μL per reaction. Techniques including quantitative PCR (qPCR) and spectrofluorimetry were used to test the purity and quantity of gDNA eluted from the chip following extraction. Bead transport and molecular diffusional analysis showed that an input of less than 4 ng of gDNA (∼667 white blood cells) is optimal for on-chip extraction. There was no observable transport of inhibitors into the eluate that would greatly affect qPCR, and a sample was successfully prepared for next-generation sequencing (NGS). The microfluidics-based extraction of DNA from whole blood described here is paramount for future work in DNA-based point-of-care diagnostics and NGS library workflows.

Mutations in Myosin 5B in Children With Early-onset Cholestasis.

Mutations in Myosin 5B (MYO5B) are known to be associated with microvillous inclusion disease (MVID) a genetic cause of neonatal intractable diarrhoea. More recently, they have been reported in children with cholestasis but without typical gastrointestinal symptoms of MVID. We describe our series of children with cholestasis and mutations in MYO5B. Clinical, laboratory, and histological data were collected from patients with cholestasis and pathogenic mutations in MYO5B, found by next generation sequencing (NGS) but with minimal gastrointestinal disease. Six patients (3 boys) were identified. Median age at presentation was 19 months (range, 3-92). Presenting features were jaundice, pale stools, pruritus, and failure to thrive. Patients 5 and 6 had intractable diarrhoea until the age of 3 and 7 years, respectively, but currently are on full enteral diet with no intestinal symptoms. Median values for serum total bilirubin were 55 μmol/L (2-500), alanine aminotransferase 73I IU/L (32-114), γ-glutamyltransferase 7 IU/L (7-10), and serum bile acids 134 μmol/L (18-274). Three patients underwent 1 or more types of biliary diversion for symptom control. Median follow-up was 5 years (2-22). At most recent follow-up, they all reported pruritus while on antipruritics. Patient 1 had a liver transplant. We identified 6 patients, with mutations in MYO5B, early-onset cholestasis and pruritus, with variable response to biliary diversion without typical MVID.

A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts.

DNA extracted from fecal samples contains DNA from the focal species, food, bacteria and pathogens. Most DNA quantification methods measure total DNA and cannot differentiate among sources. Despite the desirability of noninvasive fecal sampling for studying wildlife populations, low amounts of focal species DNA make it difficult to use for next-generation sequencing (NGS), where accurate DNA quantification is critical for normalization. Two factors are required prior to using fecal samples in NGS libraries: (1) an accurate quantification method for the amount of target DNA and (2) a determination of the relative amount of target DNA needed for successful single nucleotide polymorphism genotyping assays. Here, we address these needs by developing primers to amplify a 101 bp region of the nuclear F2 gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear (Ursus maritimus) DNA in fecal extracts. We test the assay on pure polar bear DNA extracted from muscle tissue and find a high correlation between fluorometric and qPCR quantifications. The qPCR assay was also successfully used to quantify the amount of DNA derived from polar bears in fecal extractions. Orthologs of the F2 gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies.