scholarly journals Single molecule sequencing of M13 virus genome without amplification

2017 ◽  
Author(s):  
Luyang Zhao ◽  
Liwei Deng ◽  
Gailing Li ◽  
Huan Jin ◽  
Jinsen Cai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Consensus Sequence ◽  
Virus Genome ◽  
Rna Sequences ◽  
Single Molecule Sequencing ◽  
Sequencing Platform ◽  
Single Molecule Level ◽  
M13 Virus ◽  
Third Generation Sequencing ◽  
Gc Bias

ABSTRACTThird generation sequencing is a direct measurement of DNA/RNA sequences at the single molecule level without amplification. In this study, we report sequencing of the genome of the M13 virus by a new single molecule sequencing platform. Our platform detects single molecule fluorescence by the total internal reflection microscope technique, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x and 100% coverage. The consensus sequence accuracy is 100%. We demonstrated that single molecule sequencing has no significant GC bias.

scholarly journals Resequencing theEscherichia coligenome by GenoCare single molecule sequencing platform

2017 ◽  
Author(s):  
Luyang Zhao ◽  
Liwei Deng ◽  
Gailing Li ◽  
Huan Jin ◽  
Jinsen Cai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Consensus Sequence ◽  
Illumina Miseq ◽  
Rna Sequences ◽  
Single Molecule Sequencing ◽  
Sequencing Platform ◽  
Single Molecule Level ◽  
Dna And Rna ◽  
Sequencing Platforms ◽  
Generation Sequencing

ABSTRACTNext generation sequencing (NGS) has revolutionized life sciences research. Recently, a new class of third-generation sequencing platforms has arrived to meet increasing demands in the clinic, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single molecule sequencing platform from Direct Genomics to resequence theE. coligenome and show comparable performance to the Illumina MiSeq system. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. With a consensus sequence of 99.71% nucleotide identity to that of the Illumina MiSeq system’s, GenoCare was determined to be a reliable platform for single-molecule sequencing, with strong potential for clinical applications.

scholarly journals Single molecule sequencing of the M13 virus genome without amplification

PLoS ONE ◽  
2017 ◽  
Vol 12 (12) ◽  
pp. e0188181 ◽  
Author(s):  
Luyang Zhao ◽  
Liwei Deng ◽  
Gailing Li ◽  
Huan Jin ◽  
Jinsen Cai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Virus Genome ◽  
Single Molecule Sequencing ◽  
M13 Virus

scholarly journals Accurate CNV identification from only a few cells with low GC bias in a single-molecule sequencing platform

2020 ◽  
Author(s):  
Liang Hu ◽  
Qunting Lin ◽  
Pingyuan Xie ◽  
Lidong Zeng ◽  
Lichun Liu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Single Molecule ◽  
Technical Problem ◽  
Next Generation ◽  
Single Molecule Sequencing ◽  
Sequencing Platform ◽  
Transposon Insertion ◽  
Number Variation ◽  
Gc Bias ◽  
Generation Sequencing

ABSTRACTA technical problem of characterizing copy number variation of several cells with next-generation sequencing is the whole genome amplification induced bias. The result of CNVs and mosaicism detection is affected by the GC bias. Here, we report a rapid non-WGA sample preparation strategy for a single-molecule sequencing platform GenoCare1600. This approach, combined with a single-molecule sequencing platform that avoids the use of WGA and bridge PCR processes, can provide higher reliability with its lower GC bias. By combining our optimized Tn5-based transposon insertion approach with GenoCare, we successfully detected CNVs as small as 1.29M and mosaicism as small as 20%, which is consistent with next-generation sequencing (NGS) data. Moreover, our GenoCare-TTI protocol showed less GC bias and less Mad of Diff. These results suggest that the optimized TTI approach, together with the GenoCare1600 sequencing platform, is a promising option for CNV characterization from maybe one single cell.

scholarly journals Mapping the epigenetic modifications of DNA and RNA

2020 ◽  
Vol 11 (11) ◽  
pp. 792-808 ◽  
Author(s):  
Lin-Yong Zhao ◽  
Jinghui Song ◽  
Yibin Liu ◽  
Chun-Xiao Song ◽  
Chengqi Yi
Keyword(s):  
Single Molecule ◽  
Detection Methods ◽  
Rna Modification ◽  
Rna Modifications ◽  
Single Molecule Sequencing ◽  
Dna And Rna ◽  
Third Generation Sequencing ◽  
Technological Advances ◽  
Long Read ◽  
High Throughput Detection

Abstract Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

scholarly journals Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

2012 ◽  
Vol 58 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Jessica M E van den Oever ◽  
Sahila Balkassmi ◽  
E Joanne Verweij ◽  
Maarten van Iterson ◽  
Phebe N Adama van Scheltema ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Maternal Plasma ◽  
Single Molecule Sequencing ◽  
Experimental Noise ◽  
Fetal Aneuploidy ◽  
Free Dna ◽  
Gc Bias

Abstract BACKGROUND Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. METHODS For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. RESULTS Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. CONCLUSIONS This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

scholarly journals Successful Noninvasive Trisomy 18 Detection Using Single Molecule Sequencing

2013 ◽  
Vol 59 (4) ◽  
pp. 705-709 ◽  
Author(s):  
Jessica ME van den Oever ◽  
Sahila Balkassmi ◽  
Lennart F Johansson ◽  
Phebe N Adama van Scheltema ◽  
Ron F Suijkerbuijk ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Maternal Plasma ◽  
Trisomy 18 ◽  
Diagnostic Sensitivity ◽  
Trisomy 13 ◽  
Noninvasive Detection ◽  
Single Molecule Sequencing ◽  
Gc Bias ◽  
Diagnostic Sensitivity And Specificity

BACKGROUND Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal trisomy 18 and 13 has been reported as well but seems to be less accurate with the use of this approach. The reduced accuracy can be explained by PCR-introduced guanine-cytosine (GC) bias influencing sequencing data. Previously, we demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias and result in a more pronounced noninvasive detection of fetal trisomy 21. In this study, single molecule sequencing was used for noninvasive detection of trisomy 18 and 13. METHODS Single molecule sequencing was performed on the Helicos platform with free DNA isolated from maternal plasma from 11 weeks of gestation onward (n = 17). Relative sequence tag density ratios were calculated against male control plasma samples and results were compared to those of previous karyotyping. RESULTS All trisomy 18 fetuses were identified correctly with a diagnostic sensitivity and specificity of 100%. However, low diagnostic sensitivity and specificity were observed for fetal trisomy 13 detection. CONCLUSIONS We successfully applied single molecule sequencing in combination with relative sequence tag density calculations for noninvasive trisomy 18 detection using free DNA from maternal plasma. However, noninvasive trisomy 13 detection was not accurate and seemed to be influenced by more than just GC content.

scholarly journals SMOOTH-seq: single-cell genome sequencing of human cells on a third-generation sequencing platform

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoying Fan ◽  
Cheng Yang ◽  
Wen Li ◽  
Xiuzhen Bai ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
Single Molecule ◽  
Human Cancer ◽  
Whole Genome ◽  
Third Generation ◽  
Sequencing Platform ◽  
Human Cancer Cell Lines ◽  
Third Generation Sequencing ◽  
Generation Sequencing

AbstractThere is no effective way to detect structure variations (SVs) and extra-chromosomal circular DNAs (ecDNAs) at single-cell whole-genome level. Here, we develop a novel third-generation sequencing platform-based single-cell whole-genome sequencing (scWGS) method named SMOOTH-seq (single-molecule real-time sequencing of long fragments amplified through transposon insertion). We evaluate the method for detecting CNVs, SVs, and SNVs in human cancer cell lines and a colorectal cancer sample and show that SMOOTH-seq reliably and effectively detects SVs and ecDNAs in individual cells, but shows relatively limited accuracy in detection of CNVs and SNVs. SMOOTH-seq opens a new chapter in scWGS as it generates high fidelity reads of kilobases long.

scholarly journals NanoSim: nanopore sequence read simulator based on statistical characterization

2016 ◽  
Author(s):  
Chen Yang ◽  
Justin Chu ◽  
Ren&eacute L Warren ◽  
Inanç Birol
Keyword(s):  
Single Molecule ◽  
Sequencing Technology ◽  
Statistical Characterization ◽  
Single Molecule Sequencing ◽  
Sequencing Platform ◽  
Early Access ◽  
Oxford Nanopore ◽  
Commercial Technology ◽  
Read Simulator ◽  
Oxford Nanopore Technologies

Motivation: In 2014, Oxford Nanopore Technologies (ONT) announced a new sequencing platform called MinION. The particular features of MinION reads, longer read lengths and single-molecule sequencing in particular, show potential for genome characterization. As of yet, the pre-commercial technology is exclusively available through early-access, and only a few datasets are publically available for testing. Further, no software exists that simulates MinION platform reads with genuine ONT characteristics. Results: In this article, we introduce NanoSim, a fast and scalable read simulator that captures the technology-specific features of ONT data, and allows for adjustments upon improvement of nanopore sequencing technology. Availability: NanoSim is written in Python and R. The source files and manual are available at the Genome Sciences Centre website: http://www.bcgsc.ca/platform/bioinfo/software/nanosim

scholarly journals Two Color Single Molecule Sequencing on GenoCare 1600 Platform to Facilitate Clinical Applications

2020 ◽  
Author(s):  
Fang Chen ◽  
Bin Liu ◽  
Meirong Chen ◽  
Zefei Jiang ◽  
Zhiliang Zhou ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rapid Development ◽  
Clinical Applications ◽  
Library Preparation ◽  
Sequencing Data ◽  
Single Molecule Sequencing ◽  
Sequencing Platform ◽  
E Coli ◽  
Simple Instrument ◽  
Clear Information

With the rapid development of precision medicine industry, DNA sequencing becomes increasingly important as a research and diagnosis tool. For clinical applications, medical professionals require a platform which is fast, easy to use, and presents clear information relevant to definitive diagnosis. We have developed a single molecule desktop sequencing platform, GenoCare 1600. Fast library preparation (without amplification) and simple instrument operation make it friendlier for clinical use. Here we presented sequencing data of E. coli sample from GenoCare 1600 with consensus accuracy reaches 99.99%. We also demonstrated sequencing of microbial mixtures and COVID-19 samples from throat swabs. Our data show accurate quantitation of microbial, sensitive identification of SARS-CoV-2 virus and detection of variants confirmed by Sanger sequencing.

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