scholarly journals Emulsion PCR made easy

BioTechniques ◽  
2020 ◽  
Vol 69 (1) ◽  
pp. 64-69
Author(s):  
Vaishali Verma ◽  
Amita Gupta ◽  
Vijay K Chaudhary
Keyword(s):  
Next Generation Sequencing ◽  
Gene Cloning ◽  
Organic Solvents ◽  
Dna Sequences ◽  
Dna Purification ◽  
Next Generation ◽  
Oil Droplets ◽  
Extraction Protocol ◽  
Emulsion Pcr ◽  
Generation Sequencing

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

scholarly journals Fuzzysplit: demultiplexing and trimming sequenced DNA with a declarative language

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7170
Author(s):  
Daniel Liu
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Use Cases ◽  
Next Generation ◽  
Declarative Language ◽  
Matching Accuracy ◽  
Sequencing Technologies ◽  
Preliminary Comparison ◽  
Generation Sequencing

Next-generation sequencing technologies create large, multiplexed DNA sequences that require preprocessing before any further analysis. Part of this preprocessing includes demultiplexing and trimming sequences. Although there are many existing tools that can handle these preprocessing steps, they cannot be easily extended to new sequence schematics when new pipelines are developed. We present Fuzzysplit, a tool that relies on a simple declarative language to describe the schematics of sequences, which makes it incredibly adaptable to different use cases. In this paper, we explain the matching algorithms behind Fuzzysplit and we provide a preliminary comparison of its performance with other well-established tools. Overall, we find that its matching accuracy is comparable to previous tools.

scholarly journals A systematic comparison of eight new plastome sequences from Ipomoea L

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6563
Author(s):  
Jianying Sun ◽  
Xiaofeng Dong ◽  
Qinghe Cao ◽  
Tao Xu ◽  
Mingku Zhu ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Next Generation Sequencing ◽  
Chloroplast Genome ◽  
Dna Sequences ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Next Generation ◽  
Variable Regions ◽  
Chloroplast Genomes ◽  
Generation Sequencing

Background Ipomoea is the largest genus in the family Convolvulaceae. The species in this genus have been widely used in many fields, such as agriculture, nutrition, and medicine. With the development of next-generation sequencing, more than 50 chloroplast genomes of Ipomoea species have been sequenced. However, the repeats and divergence regions in Ipomoea have not been well investigated. In the present study, we sequenced and assembled eight chloroplast genomes from sweet potato’s close wild relatives. By combining these with 32 published chloroplast genomes, we conducted a detailed comparative analysis of a broad range of Ipomoea species. Methods Eight chloroplast genomes were assembled using short DNA sequences generated by next-generation sequencing technology. By combining these chloroplast genomes with 32 other published Ipomoea chloroplast genomes downloaded from GenBank and the Oxford Research Archive, we conducted a comparative analysis of the repeat sequences and divergence regions across the Ipomoea genus. In addition, separate analyses of the Batatas group and Quamoclit group were also performed. Results The eight newly sequenced chloroplast genomes ranged from 161,225 to 161,721 bp in length and displayed the typical circular quadripartite structure, consisting of a pair of inverted repeat (IR) regions (30,798–30,910 bp each) separated by a large single copy (LSC) region (87,575–88,004 bp) and a small single copy (SSC) region (12,018–12,051 bp). The average guanine-cytosine (GC) content was approximately 40.5% in the IR region, 36.1% in the LSC region, 32.2% in the SSC regions, and 37.5% in complete sequence for all the generated plastomes. The eight chloroplast genome sequences from this study included 80 protein-coding genes, four rRNAs (rrn23, rrn16, rrn5, and rrn4.5), and 37 tRNAs. The boundaries of single copy regions and IR regions were highly conserved in the eight chloroplast genomes. In Ipomoea, 57–89 pairs of repetitive sequences and 39–64 simple sequence repeats were found. By conducting a sliding window analysis, we found six relatively high variable regions (ndhA intron, ndhH-ndhF, ndhF-rpl32, rpl32-trnL, rps16-trnQ, and ndhF) in the Ipomoea genus, eight (trnG, rpl32-trnL, ndhA intron, ndhF-rpl32, ndhH-ndhF, ccsA-ndhD, trnG-trnR, and pasA-ycf3) in the Batatas group, and eight (ndhA intron, petN-psbM, rpl32-trnL, trnG-trnR, trnK-rps16, ndhC-trnV, rps16-trnQ, and trnG) in the Quamoclit group. Our maximum-likelihood tree based on whole chloroplast genomes confirmed the phylogenetic topology reported in previous studies. Conclusions The chloroplast genome sequence and structure were highly conserved in the eight newly-sequenced Ipomoea species. Our comparative analysis included a broad range of Ipomoea chloroplast genomes, providing valuable information for Ipomoea species identification and enhancing the understanding of Ipomoea genetic resources.

scholarly journals Fuzzysplit: Demultiplexing and Trimming Sequenced DNA With a Declarative Language

2019 ◽  
Author(s):  
Daniel Liu
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Use Cases ◽  
Next Generation ◽  
Declarative Language ◽  
Matching Accuracy ◽  
Sequencing Technologies ◽  
Preliminary Comparison ◽  
Generation Sequencing

Next-generation sequencing technologies create large, multiplexed DNA sequences that require preprocessing before any further analysis. Part of this preprocessing includes demultiplexing and trimming sequences. Although there are many existing tools that can handle these preprocessing steps, they cannot be easily extended to new sequence schematics when new pipelines are developed. We present Fuzzysplit, a tool that relies on a simple declarative language to describe the schematics of sequences, which makes it incredibly adaptable to different use cases. In this paper, we explain the matching algorithms behind Fuzzysplit and we provide a preliminary comparison of its performance with other well-established tools. Overall, we find that its matching accuracy is comparable to previous tools.

scholarly journals Bacterial colonization of explanted non-endocarditis cardiac valves: evidence and characterization of the valvular microbiome

2020 ◽  
Author(s):  
Stefano Di Bella ◽  
Giuseppina Campisciano ◽  
Roberto Luzzati ◽  
Enea Gino Di Domenico ◽  
Antonio Lovecchio ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Bacterial Colonization ◽  
Common Finding ◽  
Diabetic Patients ◽  
Next Generation ◽  
Culture Methods ◽  
Bacterial Dna ◽  
Mitral Valves ◽  
Generation Sequencing

Abstract Bacterial colonization has been already demonstrated in heart valve tissues of patients without cardiovascular infections. However, the evidence of a valvular microbiome is still scarce. The next-generation sequencing method was carried out on 34 specimens of aortic (n = 20) and mitral valves (n = 14) explanted from 34 patients having neither evidence nor history of infectious diseases, particularly infective endocarditis. While no bacteria were demonstrated using standard culture methods, bacterial deoxyribonucleic acid (DNA) sequences were found using next-generation sequencing in 15/34 (44%) cases. Escherichia coli was present in 6 specimens and was the most frequently identified bacterium. There was a trend towards a higher rate of bacterial DNA positivity in specimens of calcific valves than in those of non-calcific valves (10/17 vs 5/17, P = 0.17). Based on a quantitative test, E. coli accounted for 0.7% ± 1% in calcific valvular tissue and 0.3% ± 0.3% in non-calcific valvular tissue (P = 0.2), and for 11% ± 27% in the valvular tissue of diabetic patients and 0.3% ± 1% in the valvular tissue of non-diabetic patients (P = 0.08). Detection of bacterial DNA in non-endocarditis valvular tissues could be a relatively common finding. There could be an association between the valvular microbiome and certain models of valve degeneration and common metabolic disorders.

scholarly journals BURST enables mathematically optimal short-read alignment for big data

2020 ◽  
Author(s):  
Gabriel Al-Ghalith ◽  
Dan Knights
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Heuristic Algorithms ◽  
Database Search ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Genome Database ◽  
Short Read ◽  
Lowest Common Ancestor ◽  
Generation Sequencing

AbstractOne of the fundamental tasks in analyzing next-generation sequencing data is genome database search, in which DNA sequences are compared to known reference genomes for identification or annotation. Although algorithms exist for optimal database search with perfect sensitivity and specificity, these have largely been abandoned for next-generation sequencing (NGS) data in favor of faster heuristic algorithms that sacrifice alignment quality. Virtually all DNA alignment tools that are commonly used in genomic and metagenomic database search use approximate methods that sometimes report the wrong match, and sometimes fail to find a valid match when present. Here we introduce BURST, a high-throughput DNA short-read aligner that uses several new synergistic optimizations to enable provably optimal alignment in NGS datasets. BURST finds all equally good matches in the database above a specified identity threshold and can either report all of them, pick the most likely among tied matches, or provide lowest-common-ancestor taxonomic annotation among tied matches. BURST can align, disambiguate, and assign taxonomy at a rate of 1,000,000 query sequences per minute against the RefSeq v82 representative prokaryotic genome database (5,500 microbial genomes, 19GB) at 98% identity on a 32-core computer, representing a speedup of up to 20,000-fold over current optimal gapped alignment techniques. This may have broader implications for clinical applications, strain tracking, and other situations where fast, exact, extremely sensitive alignment is desired.

scholarly journals Diagnostic Utility of Targeted Next Generation Sequencing in Patients with Vascular Anomalies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-9
Author(s):  
Whitney Eng ◽  
Sophie Dilek ◽  
Abigail Ward ◽  
Harry PW Kozakewich ◽  
Alyaa Al-Ibraheemi ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Cancer Genomics ◽  
Massively Parallel Sequencing ◽  
Genetic Diagnosis ◽  
Vascular Anomalies ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Background: Vascular anomalies are diverse entities and can range in severity from self-limiting to life-threatening. Diagnosis and care of these patients is challenging due to overlapping clinical and histologic features. Recently, it has been established that many vascular anomalies arise from somatic mutations in cancer genes (PIK3CA, AKT, NRAS). Use of cancer genomics in patients with vascular anomalies may establish a genetic diagnosis and expand use of targeted medical therapies. We evaluated the utility of targeted next generation sequencing for vascular anomalies patients at a single pediatric center. Methods: Using OncoPanel, a hybrid-capture and massively parallel sequencing assay that surveys DNA sequences of 447 genes implicated in cancer, we analyzed genetic variants in lesional tissue from vascular anomalies patients evaluated at Boston Children's Hospital between 5/2/2017 and 3/23/2020. Results: A total of 276 patients were consented and sequenced under the Dana Farber Cancer Institute Profile protocols DFCI 11-104 (n= 68) and DFCI 17-000 (n= 208). Clinical diagnoses prior to testing were varied and 11 patients (7%) had an unknown diagnosis. Tissue was analyzed for 138 patients. Targeted sequencing resulted in diagnostically significant alterations in 80 of 138 (57%) of patients and therapeutically significant alterations in 58 of 138 (42%) patients. To date, 18 patients in our cohort have been treated with medical therapy informed by their genetic diagnosis. Several more await enrollment on clinical trials. For patients with diagnoses previously categorized as unknown (n=11), sequencing led to identification of a genetic variant in 6 patients (54%). Additionally, 8/138 patients had variants requiring further evaluation for potential germline involvement. Discussion: Next generation sequencing in vascular anomalies patients identified actionable variants in a large proportion of the patients in our cohort. The mTOR inhibitor sirolimus has been used to treat a variety of vascular anomalies, but not all patients respond to this treatment. Targeted therapies based on specific genotypes hold promise as clinical trials in vascular anomalies are emerging. Additionally, sequencing in this cohort identified several variants suggesting a germline cancer predisposition requiring follow-up. Use of next generation sequencing has clinical utility and increased use of this testing may improve diagnosis, prognosis, and treatment for patients with vascular anomalies. Disclosures Adams: Novartis: Consultancy; Venthura: Consultancy. OffLabel Disclosure: Sirolimus is used off-label for the treatment of vascular anomalies.

scholarly journals Compression of Next-Generation Sequencing Data and of DNA Digital Files

Algorithms ◽  
2020 ◽  
Vol 13 (6) ◽  
pp. 151
Author(s):  
Bruno Carpentieri
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequences ◽  
Network Traffic ◽  
Large Scale ◽  
Genomic Data ◽  
Biological Data ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Generation Sequencing

The increase in memory and in network traffic used and caused by new sequenced biological data has recently deeply grown. Genomic projects such as HapMap and 1000 Genomes have contributed to the very large rise of databases and network traffic related to genomic data and to the development of new efficient technologies. The large-scale sequencing of samples of DNA has brought new attention and produced new research, and thus the interest in the scientific community for genomic data has greatly increased. In a very short time, researchers have developed hardware tools, analysis software, algorithms, private databases, and infrastructures to support the research in genomics. In this paper, we analyze different approaches for compressing digital files generated by Next-Generation Sequencing tools containing nucleotide sequences, and we discuss and evaluate the compression performance of generic compression algorithms by confronting them with a specific system designed by Jones et al. specifically for genomic file compression: Quip. Moreover, we present a simple but effective technique for the compression of DNA sequences in which we only consider the relevant DNA data and experimentally evaluate its performances.

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