scholarly journals Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

2014 ◽  
Author(s):  
Castle Raley ◽  
David Munroe ◽  
Kristie Jones ◽  
Yu-Chih Tsai ◽  
Yan Guo ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
High Input ◽  
Single Molecule Sequencing ◽  
Pacific Biosciences ◽  
Preparation Conditions ◽  
Pacbio Rs Ii ◽  
The Pacific ◽  
The Common

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms - 20 nanograms) for single-molecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

2. How to read the book of life

2018 ◽  
pp. 12-27
Author(s):  
John Archibald
Keyword(s):  
Dna Sequencing ◽  
Human Genome ◽  
Single Molecule ◽  
Human Genome Project ◽  
Genome Project ◽  
Chain Termination ◽  
Single Molecule Sequencing ◽  
Semiconductor Sequencing ◽  
The Cost ◽  
The Human Genome Project

For all its biological importance, DNA is a fragile molecule so extracting it is a difficult process. ‘How to read the book of life’ explains the techniques required to sequence DNA. It begins by explaining the techniques developed for protein and RNA sequencing by Frederick Sanger, Robert Holley, and Carl Woese that were then developed further for DNA sequencing. Following the success of the Human Genome Project, the next generation of DNA sequencing was developed in the mid-2000s. Pyrosequencing was capable of generating orders of magnitude more data at a fraction of the cost, but was superceded within a decade by semiconductor sequencing, reversible chain-termination sequencing, and single-molecule sequencing.

Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects Polyclonal and Compound BCR-ABL in Patients Who Relapse on Kinase Inhibitor Therapy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3752-3752 ◽  
Author(s):  
Catherine C. Smith ◽  
Michael Brown ◽  
Jason Chin ◽  
Corynn Kasap ◽  
Sara Salerno ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Kinase Inhibitor ◽  
Drug Resistant ◽  
Smrt Sequencing ◽  
Sequencing Technology ◽  
Employment Equity ◽  
Pacific Biosciences ◽  
Equity Ownership ◽  
Tki Treatment

Abstract Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects the Evolution of Polyclonal and Compound BCR-ABL Mutations in Patients Who Relapse On Kinase Inhibitor Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 917-917 ◽  
Author(s):  
Catherine C Smith ◽  
Michael Brown ◽  
Wendy T Parker ◽  
Kimberly Lin ◽  
Kevin Travers ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Single Molecule ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
P Value ◽  
Smrt Sequencing ◽  
Sequencing Technology ◽  
Advisory Committees ◽  
Pacific Biosciences

Abstract Abstract 917 Background: Secondary kinase domain (KD) mutations represent the most well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In CML, multiple TKIs with different mutation profiles are approved and the ability to detect KD mutations at the time of disease progression can impact therapy choice. To optimize clinical impact, second generation TKI selection must consider the majority TKI-resistant mutant population as well as smaller mutant sub-populations that may be selected with subsequent treatment. Sequential TKI therapy is associated with additional complexity: multiple mutations can coexist separately in an individual patient (“polyclonality”) or can occur in tandem on a single allele (“compound mutations”). Multiple mutations are associated with poor clinical outcome (Parker et al., Blood 2012). Compound mutations can cause in vitro resistance to ponatinib, the only TKI clinically active against the highly resistant T315I mutation (Eide, et. al, ASH 2012 abstract #1416). Currently, no clinically adaptable technology can distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD, most next-generation sequencing platforms cannot generate reads of sufficient length to determine if mutations separated by ≥500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) sequencing technology is a third generation deep sequencing technology capable of achieving average read lengths of ∼1000bp and frequently >3000bp, enabling sensitive and accurate sequencing of the entire ABL KD on a single strand of DNA. Though allele-specific detection methods such as MassARRAY offer sensitivity as low as ∼0.5%, these assays are designed to detect a limited number (∼31) of mutations whereas SMRT sequencing offers an unbiased approach capable of detecting novel variants. We sought to (1) develop a potential clinically-applicable SMRT sequencing assay for the detection of BCR-ABLKD mutations capable of distinguishing polyclonal and compound mutations, and (2) compare the accuracy and sensitivity of this method to standard sequencing and MassARRAY. Results: We assessed 54 samples from 36 CML patients who had clinically relapsed on ABL kinase inhibitor therapy and were previously analyzed by standard sequencing, and in a subset, by MassARRAY. We amplified an 863bp area of the BCR-ABLKD from patient-derived cDNA with primers containing 5' barcodes, enabling sequencing of 6 to 8 patient samples on a single SMRT cell on a single run. On average, 2519 reads were obtained for each sample per run (range 330 to 10,240). All of 131 known mutations detected by MassARRAY were identified by SMRT sequencing using a p-value threshold of 1.03e–03. SMRT sequencing also identified all 107 known mutations detected by direct sequencing with a p-value threshold of 6.0e–08. In addition to these known mutations, SMRT sequencing detected an additional 1320 non-silent mutations across all patient samples using a strict p-value threshold cut-off of 6e–08, ranging in abundance from 0.2% to 17% (median 0.75%). Among 47 samples where >1 mutation was detectable by direct sequencing or MassARRAY, SMRT sequencing revealed that 40 (85%) had compound mutations detectable at a frequency of ≥1. In total, we detected 73 different compound mutations at a frequency of ≥1%. In all cases where compound mutations were detected and more than one treatment timepoint was available, at least one compound mutation clearly evolved from a mutation detectable at a prior timepoint. In the most complex case, 4 separate mutations yielded 8 different mutant alleles. Conclusions: Pacific Biosciences RS SMRT sequencing detects KD mutations in patient samples with sensitivity comparable to or better than MassARRAY and can distinguish compound from polyclonal mutant clones. Among patient samples with multiple mutations, compound mutations were detectable in the vast majority of samples by SMRT sequencing, revealing a complex mutational landscape not demonstrable by other clinically viable sequencing methods and previously unappreciated. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles by SMRT sequencing promises to further facilitate a personalized approach to patient management and inform models of disease evolution. Disclosures: Brown: Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Shah:ARIAD, Bristol Myers-Squibb: Consultancy, Research Funding; Novartis: Consultancy.

A review of DNA sequencing techniques

2002 ◽  
Vol 35 (2) ◽  
pp. 169-200 ◽  
Author(s):  
Lilian T. C. França ◽  
Emanuel Carrilho ◽  
Tarso B. L. Kist
Keyword(s):  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Read Length ◽  
Chemical Methods ◽  
Single Molecule Sequencing ◽  
Exonuclease Digestion ◽  
Speed Accuracy ◽  
Sanger Method

1. Summary 1692. Introduction 1703. Sanger's method and other enzymic methods 1703.1 Random approach 1713.2 Direct approach 1713.3 Enzyme technology 1753.4 Sample preparation 1753.5 Labels and DNA labelling 1763.5.1 Radioisotopes 1763.5.2 Chemiluminescent detection 1763.5.3 Fluorescent dyes 1773.6 Fragment separation and analysis 1803.6.1 Electrophoresis 1803.6.2 Mass spectrometry – an alternative 1824. Maxam & Gilbert and other chemical methods 1835. Pyrosequencing – DNA sequencing in real time by the detection of released PPi 1876. Single molecule sequencing with exonuclease 1907. Conclusion 1928. Acknowledgements 1929. References 193The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the PyrosequencingTM method – DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

scholarly journals Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Yu-Chih Tsai ◽  
Sean Conlan ◽  
Clayton Deming ◽  
Julia A. Segre ◽  
Heidi H. Kong ◽  
...  
Keyword(s):  
Microbial Community ◽  
Human Skin ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
High Quality ◽  
Single Nucleotide ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
Hybrid Approaches ◽  
Long Read

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.

scholarly journals Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

2014 ◽  
Author(s):  
Konstantin Berlin ◽  
Sergey Koren ◽  
Chen-Shan Chin ◽  
James Drake ◽  
Jane M Landolin ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Locality Sensitive Hashing ◽  
Model Organisms ◽  
Smrt Sequencing ◽  
High Coverage ◽  
Celera Assembler ◽  
Single Molecule Sequencing ◽  
Long Reads ◽  
Long Read

We report reference-grade de novo assemblies of four model organisms and the human genome from single-molecule, real-time (SMRT) sequencing. Long-read SMRT sequencing is routinely used to finish microbial genomes, but the available assembly methods have not scaled well to larger genomes. Here we introduce the MinHash Alignment Process (MHAP) for efficient overlapping of noisy, long reads using probabilistic, locality-sensitive hashing. Together with Celera Assembler, MHAP was used to reconstruct the genomes of Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and human from high-coverage SMRT sequencing. The resulting assemblies include fully resolved chromosome arms and close persistent gaps in these important reference genomes, including heterochromatic and telomeric transition sequences. For D. melanogaster, MHAP achieved a 600-fold speedup relative to prior methods and a cloud computing cost of a few hundred dollars. These results demonstrate that single-molecule sequencing alone can produce near-complete eukaryotic genomes at modest cost.

scholarly journals High-coverage, long-read sequencing of Han Chinese trio reference samples

2019 ◽  
Author(s):  
Ying-Chih Wang ◽  
Nathan D Olson ◽  
Gintaras Deikus ◽  
Hardik Shah ◽  
Aaron M Wenger ◽  
...  
Keyword(s):  
Single Molecule ◽  
Han Chinese ◽  
High Coverage ◽  
Link Type ◽  
Pacific Biosciences ◽  
The Pacific ◽  
Chinese Descent ◽  
Long Read ◽  
Reference Samples

AbstractSingle-molecule long-read sequencing datasets were generated for a son-father-mother trio of Han Chinese descent that is part of the Genome In a Bottle (GIAB) consortium portfolio. The dataset was generated using the Pacific Biosciences Sequel System. The son and each parent were sequenced to an average coverage of 60 and 30, respectively, with N50 subread lengths between 16 and 18 kb. Raw reads and reads aligned to both the GRCh37 and GRCh38 are available at the NCBI GIAB ftp site (ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/) and the raw read data is archived in NCBI SRA (SRX4739017, SRX4739121, and SRX4739122). This dataset is available for anyone to develop and evaluate long-read bioinformatics methods.

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