scholarly journals Liquid drop of DNA libraries reveals total genome information

2020 ◽  
Vol 117 (44) ◽  
pp. 27300-27306 ◽  
Author(s):  
Stanislav S. Terekhov ◽  
Igor E. Eliseev ◽  
Leyla A. Ovchinnikova ◽  
Marsel R. Kabilov ◽  
Andrey D. Prjibelski ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Throughput Sequencing ◽  
Dna Templates ◽  
Single Dna Molecules ◽  
Emulsion Droplets ◽  
Dna Libraries ◽  
Emulsion Pcr ◽  
Robust Techniques ◽  
Genome Information

Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

scholarly journals Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding

MycoKeys ◽  
2018 ◽  
Vol 39 ◽  
pp. 29-40 ◽  
Author(s):  
Sten Anslan ◽  
R. Henrik Nilsson ◽  
Christian Wurzbacher ◽  
Petr Baldrian ◽  
Leho Tedersoo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Computation Time ◽  
Potential Effect ◽  
Data Sets ◽  
Sequencing Data ◽  
Operational Taxonomic Units ◽  
High Throughput Sequencing Data ◽  
Recent Developments

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.

scholarly journals DETECTION OF POTENTIALLY PATHOGENIC BACTERIA IN THE BRACKISH RIVERS FLOWING INTO THE ELTON LAKE BY HIGH-THROUGHPUT SEQUENCING

2018 ◽  
pp. 87-95 ◽  
Author(s):  
E. A. Selivanova ◽  
Yu. A. Khlopko ◽  
N. E. Gogoleva ◽  
A. O. Plotnikov
Keyword(s):  
High Throughput ◽  
Pathogenic Bacteria ◽  
Bacterial Contamination ◽  
High Throughput Sequencing ◽  
Pore Diameter ◽  
Pathogenic Microorganisms ◽  
Membrane Filters ◽  
Marine Habitats ◽  
Dna Libraries ◽  
Enzymatic Lysis

Aim. To indicate potentially pathogenic bacteria in plankton of the brackish rivers flowing into the Elton Lake by high-throughput sequencing of 16S ssuRNA gene. Materials and methods. The water samples from brackish rivers Lantsug and Chernavka, flowing into the Elton Lake, were taken up in a volume of 50 ml, filtered through membrane filters (pore diameter - 0.22 pm). Total DNAwas obtained by phenol-chloroform extraction with preliminary homogenization and enzymatic lysis. DNA libraries for sequencing were created by protocol Illumina with primers to a variable V3-V4 region of 16S ssuRNA gene. Sequencing was performed on a platform MiSeq («Illumina», США). Results.There were found the phylotypes of potentially pathogenic bacteria of Proteobacteria phylum from the families Enterobacteriaceae, Pseudomonadaceae, Campylobacteraceae, Vibrionaceae, Aeromonadaceae, Moraxellaceae, Legionellaceae, Alcaligenaceae, Campylobacteraceae, and also of Firmicutes, Bacteroidetes, Actinobacteria phyla in the plankton samples of the brackish rivers. Probable source of bacterial contamination is large and small cattle. Conclusion. These data demonstrate that the continental brackish waters, along with freshwater and marine habitats perform a reservoir function to potentially pathogenic microorganisms. High-throughput sequencing can be used to screen the presence of pathogens in water.

scholarly journals Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

2021 ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0247541
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Human Cytomegalovirus Genomes Sequenced Directly from Clinical Material: Variation, Multiple-Strain Infection, Recombination and Mutation

2018 ◽  
Author(s):  
Nicolás M. Suárez ◽  
Gavin S. Wilkie ◽  
Elias Hage ◽  
Salvatore Camiolo ◽  
Marylouisa Holton ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
High Throughput Sequencing ◽  
Clinical Pathology ◽  
Congenital Infection ◽  
Population Based ◽  
Single Strain ◽  
Natural Mutation ◽  
Future Population

ABSTRACTThe genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination and natural mutation. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analysed, in the process facilitating the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating diversity, (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination, (iii) mutants with non-functional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae, and (iv) intrahost diversity in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology and pathogenesis of HCMV.

scholarly journals Bulked Segregant Analysis Coupled With Whole-Genome Sequencing (BSA-Seq) and Identification of a Novel Locus, qGL3.5, That Regulates Grain Length

2021 ◽  
Author(s):  
Haiyan Zhao ◽  
Yuebin Zheng ◽  
Feng Bai ◽  
Yang Liu ◽  
Sicheng Deng ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bulked Segregant Analysis ◽  
Functional Characterization ◽  
Quality Data ◽  
Rice Grain ◽  
Grain Length ◽  
Starting Point ◽  
New Gene ◽  
Membrane Bound

Abstract Rice grain length (GL) directly affects the yield and quality of this species. Very few GL-related genes cloned are applied in production because their yield-increasing effect was not obvious. In this study, the two bulk-DNA pools (L-pool and S-pool) and their parents’ (KJ01 and Huaye 4) DNAs were subjected to high-throughput sequencing. After assessing the quality of the data, we obtained a total of 100.22 Gb of high-quality data; the average coverage depth was 55x, and the genome coverage was 96.51%. After combining the association results of the ED and SNP index methods, we mapped the GL genes to a 0.34 Mb “hotspot” region on chromosome 3, which contains 37 genes related to various traits. The 37 predicted genes were further analyzed by the use of the Gene GO, COG database and so on. Thirty-three genes were annotated by GO functions. According to the GO annotations, three genes whose molecular function involved in the plasma membrane and intracellular membrane-bound organelles were detected via CRISPR/ Cas9 editing technology. ORF33 was verified to regulate GL and was the target gene qGL3.5. These results provides a new gene resource for rice grain shape breeding and a starting point for functional characterization of the wild rice GL gene.

scholarly journals Somatic mutation landscapes at single-molecule resolution

2021 ◽  
Author(s):  
Stefanie V. Lensing ◽  
Peter Ellis ◽  
Federico Abascal ◽  
Iñigo Martincorena ◽  
Robert J. Osborne
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Error Rates ◽  
Dna Molecules ◽  
Base Pairs ◽  
Single Dna Molecules ◽  
Sequencing Library ◽  
Somatic Mutagenesis ◽  
Qpcr Quantification ◽  
Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Jihyun Park ◽  
Seonghyun Lee ◽  
Nabin Won ◽  
Eunji Shin ◽  
Soo-Hyun Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Physical Map ◽  
Dna Molecules ◽  
Single Dna Molecules

Two-color DNA physical map for efficient identification of single DNA molecules.

scholarly journals A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires

2020 ◽  
Vol 6 (17) ◽  
pp. eaay9093 ◽  
Author(s):  
Hidetaka Tanno ◽  
Jonathan R. McDaniel ◽  
Christopher A. Stevens ◽  
William N. Voss ◽  
Jie Li ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Low Cost ◽  
Polymerase Chain Reaction Method ◽  
Cell Receptors ◽  
Emulsion Droplets ◽  
Follicular Helper ◽  
Overlap Extension ◽  
Variable Heavy ◽  
Specialized Equipment ◽  
High Yields

Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.

scholarly journals Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss

2019 ◽  
Vol 220 (5) ◽  
pp. 781-791 ◽  
Author(s):  
Nicolás M Suárez ◽  
Gavin S Wilkie ◽  
Elias Hage ◽  
Salvatore Camiolo ◽  
Marylouisa Holton ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
High Throughput Sequencing ◽  
Gene Loss ◽  
Clinical Pathology ◽  
Congenital Infection ◽  
Population Based ◽  
Single Strain ◽  
Future Population

AbstractThe genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.

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