scholarly journals Microsatellite marker discovery using single molecule real-time circular consensus sequencing on the Pacific Biosciences RS

BioTechniques ◽  
2013 ◽  
Vol 55 (5) ◽  
Author(s):  
Markus A. Grohme ◽  
Roberto Frias Soler ◽  
Michael Wink ◽  
Marcus Frohme
Keyword(s):  
Real Time ◽  
Microsatellite Marker ◽  
Single Molecule ◽  
Pacific Biosciences ◽  
The Pacific ◽  
Marker Discovery ◽  
Circular Consensus 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.

scholarly journals Exploring DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data

2013 ◽  
Author(s):  
Sterling Sawaya ◽  
James Boocock ◽  
Mik Black ◽  
Neil Gemmell
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Double Helix ◽  
Dna Structure ◽  
R Package ◽  
Dna Structures ◽  
Pacific Biosciences ◽  
Tandem Repeat Sequences ◽  
The Pacific ◽  
Polymerase Pausing

Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA structure. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

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.

scholarly journals Pacific Biosciences Single-molecule Real-time (SMRT) Sequencing Reveals High Diversity of Basal Fungal Lineages and Stochastic Processes Controlled Fungal Community Assembly in Mangrove Sediments

2020 ◽  
Author(s):  
Zhi-Feng Zhang ◽  
Yue-Ping Pan ◽  
Yue Liu ◽  
Meng Li
Keyword(s):  
Stochastic Processes ◽  
Real Time ◽  
Community Composition ◽  
Single Molecule ◽  
Fungal Community ◽  
Community Assembly ◽  
Fungal Communities ◽  
Mangrove Sediments ◽  
Fungal Community Composition ◽  
Pacific Biosciences

Abstract Background: Mangrove wetlands are unique ecosystems with specific environmental characteristics, and are a hotspot of biodiversity. Although they probably harbor a variety of mangrove-specific fungi, the compositions of mangrove fungal community has been rarely investigated in detail, except for few published culture-based studies. In addition, the fungal community assembly and interaction patterns that impact the community composition in mangroves have not been explored to date. Results: We used the Pacific Biosciences single-molecule real-time sequencing approach targeting the entire internal transcribed spacer region, to systematically investigate the composition, biogeographical patterns, assembly processes, co-occurrence patterns and shaping factors of the fungal communities in sediments of seven representative mangroves across the Southeast China. We recovered 15 phyla, including some early diverging fungal lineages not previously reported in mangroves. Phylogenetic analysis revealed an incredibly high proportion of Rozellomycota and Chytridiomycota, as accounting for up to one-third of all fungal abundance. Although the neutral community model described a moderate portion of community variation, the similarity of fungal communities exhibited strong distance-decay patterns. Furthermore, the mean values and most beta nearest-taxon index fell between -2 to 2, with Bray–Curtis-based Raup–Crick value generally greater than 0.95, suggesting that stochastic processes strongly shape the fungal community composition. Consistently, nonmetric multidimensional scaling and permutational multivariate analysis of variance confirmed the geographical location as a crucial factor driving the distribution of both, the dominant and rare taxa of mangrove fungi. The db-RDA analyses indicated a minor role of environmental selections in shaping the fungal community. Network analyses revealed that the deep sediments harbor more complex fungal networks with highly connected taxa than surface sediments, and that rare fungal taxa might play important roles in microbial interactions and ecological functions in mangrove sediments.Conclusions: The investigation revealed high fungal diversity in mangrove sediments, with incredibly high numbers of basal fungal lineages, stochastic processes driving the assembly of fungal community, and geographic location strongly shaping fungal community composition in mangroves. These discoveries therefore spur further studies of the utilization and protection of fungal resources and communities in mangrove sediments.

scholarly journals Clustering of Circular Consensus Sequences: Accurate Error Correction and Assembly of Single Molecule Real-Time Reads from Multiplexed Amplicon Libraries

2017 ◽  
Author(s):  
Felix Francis ◽  
Michael D. Dumas ◽  
Scott B. Davis ◽  
Randall J. Wisser
Keyword(s):  
Error Correction ◽  
Real Time ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Sequencing Error ◽  
Bootstrap Analysis ◽  
Consensus Sequences ◽  
Pacific Biosciences ◽  
Local Sequence

BACKGROUNDTargeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical challenge for research and applications using HTS data, especially from long-read platforms, is errors arising from technological limits and bioinformatic algorithms.RESULTSA single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing.CONCLUSIONSC3S-LAA uses a novel processing algorithm for SMRT amplicon-sequence data that produces accurate consensus sequences and local sequence assemblies. The community standard long amplicon analysis module from Pacific Biosciences is prone to substantial errors that raise concerns about findings based on this pipeline. The method developed here removed this confounding bioinformatics source of error, allowing for the identification of limited instances of errors due to DNA amplification or sequencing.

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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