Next-generation Sequencing Libraries Research Articles

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Next-generation sequencing library construction on a surface

BackgroundNext-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface.ResultsThe surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates.ConclusionsWe described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.

Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA.

Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3′-azide-modified dideoxyribonucleotide onto the 3′-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3′-end to a 5′-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Planktonic bivalve larvae identification and quantification in Gomso Bay, South Korea, using next-generation sequencing analysis and microscopic observations

Planktonic larval stages of bivalves are notoriously difficult to identify due to their similar morphology. To evaluate changed patterns in the presented planktonic bivalve larvae within Gomso Bay (Yellow Sea, South Korea), three sites were sampled twice a month from June to October 2015 and were analysed using next-generation sequencing (NGS) and light microscopy. Bivalve larvae revealed different patterns, and at least five bivalve taxa were only detected by the NGS. According to the NGS results, most bivalve larvae were Crassostrea gigas (37%), followed by Ruditapes philippinarum (26%), and Solen strictus (23%); Mactra chinensis and M. veneriformis accounted for <1% of the bivalve larvae. C. gigas and S. strictus were most abundant in late June, but rare in the other investigated periods, while R. philippinarum was more abundant in June and late September. Microscopic observations showed a similar trend to the NGS in terms of bivalve larvae presence, although they were difficult to identify the exact species. Thus, morphological and molecular approaches should be combined in future studies to provide accurate sequences of correctly identified specimens to develop NGS libraries.

MicroRNAs associated with early neural crest development in Xenopus laevis

BackgroundThe neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression.ResultsTo uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells.ConclusionWe have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.

Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

The emergence of next-generation sequencing (NGS) over the last 10years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.

Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

Preparation of Small RNA NGS Libraries from Biofluids.

Next generation sequencing (NGS) is a powerful method for transcriptome analysis. Unlike other gene expression profiling methods, such as microarrays, NGS provides additional information such as splicing variants, sequence polymorphisms, and novel transcripts. For this reason, NGS is well suited for comprehensive profiling of the wide range of extracellular RNAs (exRNAs) in biofluids. ExRNAs are of great interest because of their possible biological role in cell-to-cell communication and for their potential use as biomarkers or for therapeutic purposes. Here, we describe a modified protocol for preparation of small RNA libraries for NGS analysis. This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications designed to reduce the sequence-specific bias typically encountered with commercial small RNA library construction kits.

Histone Native Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is becoming the standard method to study genome-wide distribution of histone variants and histone posttranslational modifications (PTMs). In this chapter, we describe a detailed native ChIP protocol and downstream procedures for the preparation of DNA libraries for next-generation sequencing. Compared to cross-linked ChIP, "native" ChIP has been shown to produce occupancy pattern data of histone PTMs and histone variants, with higher resolution and higher signal to noise ratio. We further present an adaptation of this protocol to perform native ChIP from as low as 50,000 cells.

Small RNA Isolation from Tissues of Grapevine and Woody Plants.

A protocol is described to purify small (s)RNA molecules from tissues of grapevine and other woody plants. The protocol has been specifically developed to analyze sRNA populations by high-throughput sequencing. It has been widely used on species of the genera Prunus and Vitis particularly rich in polyphenols and other enzyme-inhibiting compounds. The high quality of the sRNAs extracted from leaf or phloem tissues makes them suitable for all molecular biology reactions, in particular for next-generation sequencing library preparation.

Next-Generation Sequencing Library Preparation for 16S rRNA Microbiome Analysis After Serpin Treatment.

Serine protease inhibitors, serpins, can have profound effects on many systems in the body including immune defense systems. The microbiome, specifically the gut and lung bacterial microbiota, is now known, under some conditions, to alter immune defenses. DNA library preparation for microbiome studies is a procedure that prepares microbial genomic DNA to be sequenced in next-generation sequencing platforms. The construction involves a PCR reaction that will amplify the 16S rRNA gene and will incorporate a specific index and adaptors to the fragments. After confirmation of the product amplification by gel electrophoresis, samples will be later normalized to the same DNA amount of 240 ng. Final concentration of the library is validated by quantitative PCR (qPCR).Here we describe methods to analyze changes in the microbiome after treatment with immune-modulating agents, specifically serpins.

Small- and Large-Scale High Molecular Weight Genomic DNA Extraction from Planarians.

High-quality genomic DNA extraction is a starting point for many downstream applications in modern molecular biology. Here, we describe a simple method for isolating high molecular weight genomic DNA from planarians. The method is based on tissue lysis by a mixture of a chaotropic salt and detergent followed by organic extraction to remove proteins and lipids followed by a postpurification step to remove contaminating polysaccharides. The isolated DNA is of high molecular weight and compatible with polymerase chain reaction, cloning, or next-generation sequencing library preparation.

CleanTag Adapters Improve Small RNA Next-Generation Sequencing Library Preparation by Reducing Adapter Dimers.

Next-generation small RNA sequencing is a valuable tool which is increasing our knowledge regarding small noncoding RNAs and their function in regulating genetic information. Library preparation protocols for small RNA have thus far been restricted due to higher RNA input requirements (>10ng), long workflows, and tedious manual gel purifications. Small RNA library preparation methods focus largely on the prevention or depletion of a side product known as adapter dimer that tends to dominate the reaction. Adapter dimer is the ligation of two adapters to one another without an intervening library RNA insert or any useful sequencing information. The amplification of this side reaction is favored over the amplification of tagged library since it is shorter. The small size discrepancy between these two species makes separation and purification of the tagged library very difficult. Adapter dimer hinders the use of low input samples and the ability to automate the workflow so we introduce an improved library preparation protocol which uses chemically modified adapters (CleanTag) to significantly reduce the adapter dimer. CleanTag small RNA library preparation workflow decreases adapter dimer to allow for ultra-low input samples (down to approx. 10pg total RNA), elimination of the gel purification step, and automation. We demonstrate how to carry out this streamlined protocol to improve NGS data quality and allow for the use of sample types with limited RNA material.

ClickSeq: Replacing Fragmentation and Enzymatic Ligation with Click-Chemistry to Prevent Sequence Chimeras.

We recently reported a fragmentation-free method for the synthesis of Next-Generation Sequencing libraries called "ClickSeq" that uses biorthogonal click-chemistry in place of enzymes for the ligation of sequencing adaptors. We found that this approach dramatically reduces artifactual chimera formation, allowing the study of rare recombination events that include viral replication intermediates and defective-interfering viral RNAs. ClickSeq illustrates how robust, bio-orthogonal chemistry can be harnessed in vitro to capture and dissect complex biological processes. Here, we describe an updated protocol for the synthesis of "ClickSeq" libraries.

Next-Generation Sequencing of Genome-Wide CRISPR Screens.

Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Comparison of HLA DQA1 and DQB1 Genes by Multiplex Ligation-Dependent Probe Amplification and Next Generation Sequencing Technologies for Celiac Disease Risk Determination

Introduction: Celiac disease is a systemic autoimmune disease with chronic gastrointestinal disorder caused by intolerance to gluten proteins. The most dominant genetic risk factors are the specific genotypes encoding the Human Leukocyte Antigen (HLA) class II molecule genes, DQA1 and DQB1. The DQ2.5 variant (DQA1*05/DQB1*02 alleles) is present in 90-95% of patients with celiac disease. The DQ8 variant (DQA1*03/DQB1*0302) and DQ2.2 variant (DQA1*02/DQB1*02) are found in most of the remaining patients. A positive test is indicative of genetic susceptibility but does not necessarily mean disease development. A negative test has a more significant value because gluten intolerance rarely occurs in the absence of specific HLA predisposing alleles. In the current study we compared Multiplex Ligation-dependent Probe Amplification (MLPA) and Next Generation Sequencing (NGS) technologies for identifying Celiac disease risk HLA alleles. Methods: DNA was extracted from either saliva or whole-blood archival samples obtained from commercial vendors using Qiagen DNA mini extraction kits. MLPA assay probe mix designed to detect the HLA alleles implicated in Celiac disease consists of 29 specific fluorescent labeled amplification products. The PCR products are separated by capillary gel electrophoresis on the ABI 3500 Genetic Analyzer yielding a specific electropherogram. Based on the presence and relative height of individual probe, as compared to the relative probe peak height in various reference samples, the HLA allele in the patient sample was determined. For Next Generation sequencing assay, long range PCR products of HLA DQA1 and DQB1 genes were pooled to prepare bar coded NGS libraries. Equimolar pools of the barcoded libraries were sequenced on the Illumina MiSeq platform by paired end 151 bp sequencing. Demultiplexed FASTQ files were aligned to HLA variant database and genotypes were identified. Results: A total of 26 samples with different HLA variants were analyzed by both MLPA and NGS assay panel. The samples included DQ2.5, DQ8, DQ2.2, both heterozygous and homozygous variants, negatives and positive control cell line DNA samples. Data is summarized in Table 1. There was 100% concordance between the two technologies in determining the Celiac disease risk alleles. DNA dilution studies showed that both technologies were optimal with a minimum of 40 ng of DNA input. The workflow for MLPA was simple with sample to data generation completed within 24 hours. NGS assay was laborious with highly technical library preparation, sequencing and data analysis with sample to data in 48 hours. However, NGS technology results in a high resolution genotyping whereas MLPA assay is limited to 5 alleles of DQA1 and DQB1 genes.Table: Table. HLA DQA1 and DQB1 genotyping data summary- MLPA and NGS dataConclusion: Both methods are reliable and efficient to provide necessary information for celiac disease risk evaluation. In the future if high resolution HLA genotyping is needed, NGS may be more applicable.

The unexpected depths of genome-skimming data: A case study examining Goodeniaceae floral symmetry genes.

Premise of the study:The use of genome skimming allows systematists to quickly generate large data sets, particularly of sequences in high abundance (e.g., plastomes); however, researchers may be overlooking data in low abundance that could be used for phylogenetic or evo-devo studies. Here, we present a bioinformatics approach that explores the low-abundance portion of genome-skimming next-generation sequencing libraries in the fan-flowered Goodeniaceae.Methods:Twenty-four previously constructed Goodeniaceae genome-skimming Illumina libraries were examined for their utility in mining low-copy nuclear genes involved in floral symmetry, specifically the CYCLOIDEA (CYC)-like genes. De novo assemblies were generated using multiple assemblers, and BLAST searches were performed for CYC1, CYC2, and CYC3 genes.Results:Overall Trinity, SOAPdenovo-Trans, and SOAPdenovo implementing lower k-mer values uncovered the most data, although no assembler consistently outperformed the others. Using SOAPdenovo-Trans across all 24 data sets, we recovered four CYC-like gene groups (CYC1, CYC2, CYC3A, and CYC3B) from a majority of the species. Alignments of the fragments included the entire coding sequence as well as upstream and downstream regions.Discussion:Genome-skimming data sets can provide a significant source of low-copy nuclear gene sequence data that may be used for multiple downstream applications.