Next-generation Sequencing Libraries Research Articles

Presence and distribution of insect-associated and entomopathogenic fungi in a temperate pine forest soil: An integrated approach

For decades entomopathogenic fungi have garnered interest as possible alternatives to chemical pesticides. However, their ecology outside of agroecosystems demands further study. We assessed the diversity and abundance of entomopathogenic and insect-associated fungi at a loblolly pine forest in North Carolina, USA using culture-dependent and next-generation sequencing libraries. Fungi were isolated using Galleriamellonella larvae, as well as from soil dilutions plated on a selective medium. Isolates were identified using Sanger sequencing of the ITS and LSU rRNA gene regions, and represented 36 OTUs including Metarhizium, Lecanicillium, and Paecilomyces. Additionally, we assessed the chitinolytic potential of isolates and found widespread, variable ability to degrade chitin within and between genera. Phylogenetic analyses resolved several isolates to genus, with some forming clades with other insect-associated taxa, as well as with fungi associated with plant tissues. Saprophytes were widely distributed in soil, while entomopathogens were less abundant and present primarily in the top two cm of the soil. The similarity between culture-dependent and next-generation sequencing results demonstrates that both methods can be used concurrently in this system to study the ecology of entomopathogenic fungi.

Comparison of seven methods for DNA extraction from prosomata of the acorn barnacle, Amphibalanus amphitrite

Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

BackgroundDetection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine.MethodsWe selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples.ResultsAll UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment.ConclusionIncluding UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Abstract 3522: Enzymatic DNA repair enables high quality library preparation and accurate sequencing from highly damaged DNA inputs

Abstract High-throughput sequencing is key for clinical diagnostic applications such as disease mutation profiling and identifying pathogens with high taxonomic specificity. Preparation of high quality next-generation sequencing (NGS) libraries is critical for obtaining accurate patient data, but clinical scientists are often limited by input DNA quality. In cancer genomics, formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy is a common source of DNA, but the extracted DNA is often poor in both yield and quality due to fixation- and storage-induced damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-related variants from noise. Even relatively high-quality DNA inputs contain nicks and gaps limiting library yields and read lengths for long read sequencing platforms including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio). We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries through efficient nick and gap repair. Illumina library yield and quality were improved when DNA samples were treated with FFPE DNA Repair V2 before library preparation. Target enrichment and whole exome enrichment experiments showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls due to damage-induced sequence artifacts. True mutation frequencies were validated using droplet digital PCR (ddPCR). Repaired ONT libraries using DNA inputs of varying quality also showed improved library quality as well as greater read lengths. Therefore, enzymatic DNA repair prior to NGS library preparation is a critical first step for improving the quality and accuracy of short- and long-read sequencing libraries. Citation Format: Margaret R. Heider, Lixin Chen, Chen Song, Luo Sun, Pingfang Liu, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. Enzymatic DNA repair enables high quality library preparation and accurate sequencing from highly damaged DNA inputs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3522.

Abstract 439: Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells

Abstract Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~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 elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel. Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite. Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was &amp;lt; 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis. Conclusions. Amplicon-based targeted single-cell 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. Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.

Abstract 3007: Validation of an NGS panel assay for detection of hotspot cancer somatic mutations

Abstract Background The gene tests to predict whether cancers respond to specific targeted therapies or prediction of prognosis has quickly increased over the last decade. Next-generation sequencing (NGS) has become a powerful tool for high-throughput and sensitive gene mutation testing, which is required for development of targeted therapies, clinical trials, and further research. The aim of this study was to validate the AmpliSeq for Illumina Cancer Hotspot Panel v2 on the Illumina MiSeq platform and simultaneously analyze 2800 somatic mutations across the hotspot regions of 50 genes, including SNP and small INDEL, with known associations to cancer, as identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. This panel enables the study of the genes associated with different cancer types, including the cancers of the lung, colon, breast, thyroid, oral, bladder, prostate, liver, kidney, ovarian and cervix, etc. Methods and findings We validated this assay using the genomic DNA extracted from human paraffin-embedded (FFPE) samples and peripheral blood with negative control DNA (NA12878), positive control DNA (HD701), blank control as well as the global proficiency testing samples. Following two lab scientists parallel NGS library preparation, sequenced on Miseq platform (2x150 PE) and analyzed using a pipeline centered on Illumina Basespace, the somatic mutations were called at an average number of reads coverage &amp;gt;2500X, variant frequency cutoff 1%. An integrated test report can be generated within five working days. Panel sequencing analysis showed that the mutations are 100% concordance with known mutations and consistent in four runs, between lab personal performances, different input DNA concentrations (1ng-100ng), etc. Briefly, 25 mutations were detected in the FFPE samples and 20 mutations detected in proficiency testing samples. The variant types, HGVS ID and frequency are 100% match with its standard results which are a prove of precision and sensitivity. Conclusions A sensitive, high-throughput, pan-cancer mutation panel for NGS analysis of cancer hot-spot mutations in 50 genes was highly repeatable and reproducible and validated for routine application in a gene testing lab. Note: This abstract was not presented at the meeting. Citation Format: Wenzhi Li, Henry She, Xiaohua Tian, Margaret Neja, Adrian Harris, Yuhua Li. Validation of an NGS panel assay for detection of hotspot cancer somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3007.

Abstract CT143: Baseline circulating tumor DNA (ctDNA) analysis in Asian women with HR+/HER2- advanced breast cancer (ABC) receiving ribociclib + endocrine therapy in the Phase Ib MONALEESASIA trial

Abstract Background: MONALEESASIA (NCT02333370) is an ongoing Phase Ib study in which Asian patients (pts) with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) ABC are treated with ribociclib (a cyclin-dependent kinase 4/6 inhibitor) and either letrozole (all pts), fulvestrant, or tamoxifen (Japanese pts). Tumor biomarker analysis in MONALEESASIA using NanoString technology for gene expression was presented previously (Yap YS, et al. ESMO Asia 2018 [Abstract 400]). Here we report additional biomarker analysis of baseline ctDNA. Methods: Plasma samples were collected at baseline and end of treatment; ctDNA was extracted and used to generate next-generation sequencing libraries. The DNA libraries were then enriched for a specific 2.9 Mb of the human genome containing ≈550 oncogenes and tumor suppressor genes. Captured libraries were then combined in equimolar pools and sequenced, targeting ≥70 million sequencing reads per sample to ensure that unique coverage of the genes was &amp;gt;1000× or approached the maximal complexity of the library. Results: ctDNA was analyzed in baseline plasma samples collected from 77 pts (49 Japanese and 28 non-Japanese). Similar gene alteration frequencies were observed in Japanese and non-Japanese pts except for ESR1 alterations, which were more common in Japanese pts (Table). TP53 alterations were more common in pts with progressive disease, and the frequencies of TP53, ESR1, and FAT1 were slightly lower in pre- vs postmenopausal pts; however, these differences were not statistically significant. Response rates by genetic alteration are shown in the table. Conclusion: There appeared to be a trend toward a higher likelihood of alterations in TP53 and PIK3CA in pts with poorer responses, although the sample size was small. These data are hypothesis generating, and additional analysis is ongoing. Gene AlterationsPIK3CATP53CCND1FAT1ESR1FGFR1CDH1Frequency, n (%)Japanesen=4917 (35)11 (22)5 (10)3 (6)7 (14)5 (10)4 (8)Non-Japanesen=288 (29)7 (25)3 (11)5 (18)02 (7)0Best Overall Response, n of Patients with Alteration (% Response Type)PRn=359 (26)6 (17)5 (14)3 (9)2 (6)4 (11)0SDn=299 (31)7 (24)1 (3)3 (10)3 (10)03 (10)NCRNPDn=63 (50)1 (17)002 (33)1 (17)1 (17)PDn=74 (57)4 (57)2 (29)2 (29)02 (29)0CCND1, cyclin D1; CDH1, cadherin 1; ESR1, estrogen receptor 1; FAT1, FAT atypical cadherin 1; FGFR1, fibroblast growth factor receptor 1; NCRNPD, not complete response nor progressive disease; PD, progressive disease; PIK3CA, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α; PR, partial response; SD, stable disease; TP53, tumor protein p53. Citation Format: Yoon-Sim Yap, Faye Su, Nadia Solovieff, Yoshinori Ito, Norikaza Masuda, Takashi Ishikawa, Tomoyuki Aruga, Seung Jin Kim, Wei He, Mihaela Gazdoiu, Joanne Chiu. Baseline circulating tumor DNA (ctDNA) analysis in Asian women with HR+/HER2- advanced breast cancer (ABC) receiving ribociclib + endocrine therapy in the Phase Ib MONALEESASIA trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT143.

Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue.

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

Shedding Light on a Secretive Tertiary urodelean Relict: Hynobiid salamanders (Paradactylodon persicus s.l.) from Iran, Illuminated by Phylogeographic, Developmental and Transcriptomic Data.

The Hyrcanian Forests present a unique Tertiary relict ecosystem, covering the northern Elburz and Talysh Ranges (Iran, Azerbaijan), a poorly investigated, unique biodiversity hotspot with many cryptic species. Since the 1970s, two nominal species of Urodela, Hynobiidae, Batrachuperus (later: Paradactylodon) have been described: Paradactylodon persicus from northwestern and P. gorganensis from northeastern Iran. Although P. gorganensis has been involved in studies on phylogeny and development, there is little data on the phylogeography, systematics, and development of the genus throughout the Hyrcanian Forests; genome-wide resources have been entirely missing. Given the huge genome size of hynobiids, making whole genome sequencing hardly affordable, we aimed to publish the first transcriptomic resources for Paradactylodon from an embryo and a larva (9.17 Gb RNA sequences; assembled to 78,918 unigenes). We also listed 32 genes involved in vertebrate sexual development and sex determination. Photographic documentation of the development from egg sacs across several embryonal and larval stages until metamorphosis enabled, for the first time, comparison of the ontogeny with that of other hynobiids and new histological and transcriptomic insights into early gonads and timing of their differentiation. Transcriptomes from central Elburz, next-generation sequencing (NGS) libraries of archival DNA of topotypic P. persicus, and GenBank-sequences of eastern P. gorganensis allowed phylogenetic analysis with three mitochondrial genomes, supplemented by PCR-amplified mtDNA-fragments from 17 museum specimens, documenting <2% uncorrected intraspecific genetic distance. Our data suggest that these rare salamanders belong to a single species P. persicus s.l. Humankind has a great responsibility to protect this species and the unique biodiversity of the Hyrcanian Forest ecosystems.

A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5′ and 3′ termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

The study of rotavirus phylogenetic diversity, re-assortment and interspecies transmission and associated virome in Northern Irish livestock and wildlife

Background Rotavirus (RV) is a highly infectious pathogen of livestock causing diarrhoea and dehydration, and substantial economic loss. RV is an RNA virus with a segmented genome that has evolved significantly creating diverse strains. RV is believed to be endemic within livestock and the genotyping of positive isolates will allow the diversity of the rotavirus to be phylogenetically mapped for evidence of re-assortment and interspecies transmission. Rotavirus positive samples and rotavirus negative samples were examined using a metagenomics approach through next generation sequencing (NGS) to study the virome of symptomatic animals. Methods RV in symptomatic livestock, and a small subset of wildlife and exotic animal faeces samples (n=326), originating from Northern Ireland, were screened by RT PCR for the RV VP6 gene. NGS libraries using a de novo metagenomics approach were run on the MiSeq reagent V3 600 cycle as pairend reads. Data was quality checked by Fast QC, assembled in SPAdes, processed through NCBI blast (n) and then MEGAN to display the taxonomical content. Results The prevalence of RV VP6 gene was n=108 (33 %). Initial Sanger sequencing results showed porcine G3, G4 and G5 and bovine P7 and P13 strains. Preliminary metagenomic results indicates re-assortment and interspecies transmission with a positive bovine sample showing RV acquired re-assortment from human and equine RV strains. Analysis shows there is a large viral community present in both RV positive and RV negative animals. The metagenomic profiling through NGS data set will give a better understanding of the symptomatic virome in livestock.

Decoding genetic and epigenetic information embedded in cell free DNA with adapted SALP-seq.

Cell free DNA (cfDNA) in human plasma carries abundant information, which has therefore been the key sample for noninvasive prenatal testing (NIPT) and liquid biopsy. Especially by using the rapidly developed next-generation sequencing (NGS) techniques, the genetic and epigenetic information embedded in cfDNA has been effectively and extensively decoded. In this process, a key step is to construct the NGS library. Due to its high degradation, the single strand-based method was reported to be more qualified to construct the NGS library of cfDNA. In order to develop a new simple method for this application, this study adapted our recently developed single strand adaptor library preparation (SALP) method for constructing an NGS library of cfDNA. In the improved method, cfDNA was firstly denatured into single strands and then ligated with a single strand adaptor (SSA) that had a 3' overhang of 3 random bases by using T4 DNA ligase. The SSA-ligated DNA was converted into double-stranded DNA with an additional adenine at the other end by polymerizing with Taq polymerase. Next, a barcode T adaptor (BTA) was ligated to this end. Finally, the cfDNA ligated with two adaptors was amplified with the Illumina-compatible primers for NGS. Using the method, this study successfully sequenced 20 cfDNA samples from 16 esophageal cancer patients and 4 healthy people. By bioinformatics analysis, this study found the genetic and epigenetic difference between patients and healthy people and identified 23 epigenetic and 28 genetic altered esophageal cancer-specific genes.

Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies

The emergence of highly sensitive molecular diagnostic approaches, such as droplet digital PCR, has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytic strategies that integrate molecular barcodes have rarely been applied to clinical settings. In this study, we evaluated the parameters that are crucial in the use of molecular barcodes in next-generation sequencing for genotyping clinical specimens from patients with hematologic malignancies. The uniform incorporation of molecular barcodes into DNA templates through PCR was found to be crucial, and the extent of uniformity was governed by multiple interdependent variables. An error elimination strategy was developed for removing sequencing background errors by using molecular barcode sequence information as an alternative to the conventional error correction approach. This approach was successfully used to identify mutations with frequencies as low as 0.15%, and the clonal heterogeneity of hematologic malignancies was revealed. These findings have implications for elucidating heterogeneity and temporal and spatial clonal evolution, evaluating response to therapy, and monitoring relapse in patients with hematologic malignancies.

Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon.

Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.

Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody™ Single-Cell Analysis System.

In this review, we describe the BD Rhapsody™ Single-Cell Analysis System, a platform that allows high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and a multitier barcoding system. The resulting captured information can be used to generate various types of next-generation sequencing (NGS) libraries, including whole transcriptome analysis for discovery biology and targeted RNA analysis for high sensitivity transcript detection. The BD Rhapsody system can be used with emerging applications, such as BD™ AbSeq assays, to profile gene expression in both mRNA and protein level to provide ultra-high resolution analysis of single cells.

NGS-Based High-Throughput Screen to Identify MicroRNAs Regulating Growth of B-Cell Lymphoma.

MicroRNAs (miRNAs) play important roles in development, differentiation, and homeostasis by regulating protein translation. In B-cell lymphoma, many miRNAs have altered expression levels, and for a limited subset of them, experimental data supports their functional relevance in lymphoma pathogenesis. This chapter describes an unbiased next-generation sequencing (NGS)-based high-throughput screening approach to identify miRNAs that are involved in the control of cell growth. First, we provide a protocol for performing high-throughput screening for miRNA inhibition and overexpression. Second, we describe the procedure for next-generation sequencing library preparation. Third, we provide a workflow for data analysis.

Whole genome sequencing of Indonesian dengue virus isolates using next-generation sequencing

Indonesia is a tropical country and hyperendemic for dengue. The disease prevalently affected Indonesian and it caused high morbidity and substantial economic burden. This vector-borne viral disease is caused by infection of dengue viruses (DENVs), which are the member of Flaviviridae family. While most of dengue studies in Indonesia focused on the epidemiology, the clinical aspects, the vectors, and to certain extent the virology, there were still gaps in the DENVs genomic aspects. Considering their high mutation rate, the DENVs were known for their high genetic diversity and it might affect the characteristics of the viruses. Comprehensive DENV genomic data were thus important for many aspects of disease management, including virus surveillance, pathogenesis, diagnostics, antiviral drug design, and vaccine development. We established in this study a method for DENV whole genome sequencing using the advanced Next-Generation Sequencing (NGS) and Nextera XT DNA library preparation kit, coupled with simplified bioinformatic analysis methods. The Indonesian DENVs from four serotypes were isolated from patients’ sera, while library was prepared from enriched templates and sequenced using Illumina NGS. Our study highlighted the potential of a robust NGS method in producing whole genome sequence of DENVs, which would be important for future dengue studies.

Enhanced bioinformatic profiling of VIDISCA libraries for virus detection and discovery

VIDISCA is a next-generation sequencing (NGS) library preparation method designed to enrich viral nucleic acids from samples before highly-multiplexed low depth sequencing. Reliable detection of known viruses and discovery of novel divergent viruses from NGS data require dedicated analysis tools that are both sensitive and accurate. Existing software was utilised to design a new bioinformatic workflow for high-throughput detection and discovery of viruses from VIDISCA data. The workflow leverages the VIDISCA library preparation molecular biology, specifically the use of Mse1 restriction enzyme which produces biological replicate library inserts from identical genomes. The workflow performs total metagenomic analysis for classification of non-viral sequence including parasites and host, and separately carries out virus specific analyses. Ribosomal RNA sequence is removed to increase downstream analysis speed and remaining reads are clustered at 100% identity. Known and novel viruses are sensitively detected via alignment to a virus-only protein database, and false positives are removed. A new cluster-profiling analysis takes advantage of the viral biological replicates produced by Mse1 digestion, using read clustering to flag the presence of short genomes at very high copy number. Importantly, this analysis ensures that highly repeated sequences are identified even if no homology is detected, as is shown here with the detection of a novel gokushovirus genome from human faecal matter. The workflow was validated using read data derived from serum and faeces samples taken from HIV-1 positive adults, and serum samples from pigs that were infected with atypical porcine pestivirus.

SMARTcleaner: identify and clean off-target signals in SMART ChIP-seq analysis

BackgroundNoises and artifacts may arise in several steps of the next-generation sequencing (NGS) process. Recently, an NGS library preparation method called SMART, or Switching Mechanism At the 5′ end of the RNA Transcript, is introduced to prepare ChIP-seq (chromatin immunoprecipitation and deep sequencing) libraries from small amount of DNA material, using the DNA SMART ChIP-seq Kit. The protocol adds Ts to the 3′ end of DNA templates, which is subsequently recognized and used by SMART poly(dA) primers for reverse transcription and then addition of PCR primers and sequencing adapters. The poly(dA) primers, however, can anneal to poly(T) sequences in a genome and amplify DNA fragments that are not enriched in the immunoprecipitated DNA templates. This off-target amplification results in false signals in the ChIP-seq data.ResultsHere, we show that the off-target ChIP-seq reads derived from false amplification of poly(T/A) genomic sequences have unique and strand-specific features. Accordingly, we develop a tool (called “SMARTcleaner”) that can exploit these features to remove SMART ChIP-seq artifacts. Application of SMARTcleaner to several SMART ChIP-seq datasets demonstrates that it can remove reads from off-target amplification effectively, leading to significantly improved ChIP-seq peaks and results.ConclusionsSMARTcleaner could identify and clean the false signals in SMART-based ChIP-seq libraries, leading to improvement in peak calling, and downstream data analysis and interpretation.