June 1, 2021  |  

Evaluating the potential of new sequencing technologies for genotyping and variation discovery in human data.

A first look at Pacific Biosciences RS data Pacific Biosciences technology provides a fundamentally new data type that provides the potential to overcome these limitations by providing significantly longer reads (now averaging >1kb), enabling more unique seeds for reference alignment. In addition, the lack of amplification in the library construction step avoids a common source of base composition bias. With these potential advantages in mind, we here evaluate the utility of the Pacific Biosciences RS platform for human medical resequencing projects by assessing the quality of the raw sequencing data, as well as its use for SNP discovery and genotyping using the Genome Analysis Toolkit (GATK).


June 1, 2021  |  

Single Molecule Real-Time (SMRT) Sequencing of genes implicated in autosomal recessive diseases.

In today’s clinical diagnostic laboratories, the detection of the disease causing mutations is either done through genotyping or Sanger sequencing. Whether done singly or in a multiplex assay, genotyping works only if the exact molecular change is known. Sanger sequencing is the gold standard method that captures both known and novel molecular changes in the disease gene of interest. Most clinical Sanger sequencing assays involve PCR-amplifying the coding sequences of the disease target gene followed by bi-directional sequencing of the amplified products. Therefore for every patient sample, one generates multiple amplicons singly and each amplicon leads to two separate sequencing reactions. Single Molecule, Real-Time (SMRT) sequencing offers several advantages to Sanger sequencing including long read lengths, first-in-first-out processing, fast time to result, high-levels of multiplexing and substantially reduced costs. For our first proof-of-concept experiment, we queried 3 known disease-associated mutations in de-identified clinical samples. We started off with 3 autosomal recessive diseases found at an increased frequency in the Ashkenazi Jewish population: Tay Sachs disease, Niemann-Pick disease and Canavan disease. The mutated gene in Tays Sachs is HEXA, Niemann-Pick is SMPD1 and Canavan is ASPA. Coding exons were amplified in multiple (6-13) amplicons for each gene from both non-carrier and carriers. Amplicons were purified, concentrations normalized, and combined prior to SMRTbell™ Library prep. A single SMRTbell library was sequenced for each gene from each patient using standard Pacific Biosciences C2 chemistry and protocols. Average read lengths of 4,000 bp across samples allowed for high-quality Circular Consensus Sequences (CCS) across all amplicons (all less than 1 kb). This high quality CCS data permitted the clean partitioning of reads from a patient in the presence of heterozygous events. Using non-carrier sequencing as a control, we were able to correctly identify the known events in carrier genes. This suggests the potential utility of SMRT sequencing in a clinical setting, enabling a cost-effective method of replacing targeted mutation detection with sequencing of the entire gene.


June 1, 2021  |  

Making the most of long reads: towards efficient assemblers for reference quality, de novo reconstructions

2015 SMRT Informatics Developers Conference Presentation Slides: Gene Myers, Ph.D., Founding Director, Systems Biology Center, Max Planck Institute delivered the keynote presentation. He talked about building efficient assemblers, the importance of random error distribution in sequencing data, and resolving tricky repeats with very long reads. He also encouraged developers to release assembly modules openly, and noted that data should be straightforward to parse since sharing data interfaces is easier than sharing software interfaces.


June 1, 2021  |  

Un-zipping diploid genomes – revealing all kinds of heterozygous variants from comprehensive hapltotig assemblies

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON (https://github.com/PacificBiosciences/FALCON), we developed new algorithms and software (FALCON-unzip) for de novo haplotype reconstructions from SMRT Sequencing data. We apply the algorithms and the prototype software for (1) a highly repetitive diploid fungal genome (Clavicorona pyxidata) and (2) an F1 hybrid from two inbred Arabidopsis strains: CVI-0 and COL-0. For the fungal genome, we achieved an N50 of 1.53 Mb (of the 1n assembly contigs) of the ~42 Mb 1n genome and an N50 of the haplotigs of 872 kb from a 95X read length N50 ~16 kb dataset. We found that ~ 45% of the genome was highly heterozygous and ~55% of the genome was highly homozygous. We developed methods to assess the base-level accuracy and local haplotype phasing accuracy of the assembly with short-read data from the Illumina platform. For the Arabidopsis F1 hybrid genome, we found that 80% of the genome could be separated into haplotigs. The long range accuracy of phasing haplotigs was evaluated by comparing them to the assemblies from the two inbred parental lines. We show that a more complete view of all haplotypes could provide useful biological insights through improved annotation, characterization of heterozygous variants of all sizes, and resolution of differential allele expression. Finally, we applied this method to WGS human data sets to demonstrate the potential for resolving complicated, medically-relevant genomic regions.


April 21, 2020  |  

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Long-read RNA sequencing (RNA-seq) is promising to transcriptomics studies, however, the alignment of the reads is still a fundamental but non-trivial task due to the sequencing errors and complicated gene structures. We propose deSALT, a tailored two-pass long RNA-seq read alignment approach, which constructs graph-based alignment skeletons to sensitively infer exons, and use them to generate spliced reference sequence to produce refined alignments. deSALT addresses several difficult issues, such as small exons, serious sequencing errors and consensus spliced alignment. Benchmarks demonstrate that this approach has a better ability to produce high-quality full-length alignments, which has enormous potentials to transcriptomics studies.


April 21, 2020  |  

A comprehensive evaluation of long read error correction methods

Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods, and provide a comprehensive evaluation of the corresponding long read error correction tools. Leveraging recent real sequencing data, we establish benchmark data sets and set up evaluation criteria for a comparative assessment which includes quality of error correction as well as run-time and memory usage. We study how trimming and long read sequencing depth affect error correction in terms of length distribution and genome coverage post-correction, and the impact of error correction performance on an important application of long reads, genome assembly. We provide guidelines for practitioners for choosing among the available error correction tools and identify directions for future research.


April 21, 2020  |  

Fast and accurate long-read assembly with wtdbg2

Existing long-read assemblers require tens of thousands of CPU hours to assemble a human genome and are being outpaced by sequencing technologies in terms of both throughput and cost. We developed a novel long-read assembler wtdbg2 that, for human data, is tens of times faster than published tools while achieving comparable contiguity and accuracy. It represents a significant algorithmic advance and paves the way for population-scale long-read assembly in future.


April 21, 2020  |  

Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.


April 21, 2020  |  

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Efficiency of PacBio long read correction by 2nd generation Illumina sequencing.

Long sequencing reads offer unprecedented opportunities in analysis and reconstruction of complex genomic regions. However, the gain in sequence length is often traded for quality. Therefore, recently several approaches have been proposed (e.g. higher sequencing coverage, hybrid assembly or sequence correction) to enhance the quality of long sequencing reads. A simple and cost-effective approach includes use of the high quality 2nd generation sequencing data to improve the quality of long reads. We designed a dedicated testing procedure and selected universal programs for long read correction, which provide as the output sequences that can be used in further genomic and transcriptomic studies. Our results show that HALC is the best choice for correction of long PacBio reads, when both, read size and quality, are the main focus of the analysis. However, the tested tools show some unexpected behaviors, including read trimming and fragmentation.Copyright © 2017 Elsevier Inc. All rights reserved.


April 21, 2020  |  

Novel trimethoprim resistance gene dfrA34 identified in Salmonella Heidelberg in the USA.

Trimethoprim/sulfamethoxazole is a synthetic antibiotic combination recommended for the treatment of complicated non-typhoidal Salmonella infections in humans. Resistance to trimethoprim/sulfamethoxazole is mediated by the acquisition of mobile genes, requiring both a dfr gene (trimethoprim resistance) and a sul gene (sulfamethoxazole resistance) for a clinical resistance phenotype (MIC =4/76?mg/L). In 2017, the CDC investigated a multistate outbreak caused by a Salmonella enterica serotype Heidelberg strain with trimethoprim/sulfamethoxazole resistance, in which sul genes but no known dfr genes were detected.To characterize and describe the molecular mechanism of trimethoprim resistance in a Salmonella Heidelberg outbreak isolate.Illumina sequencing data for one outbreak isolate revealed a 588?bp ORF encoding a putative dfr gene. This gene was cloned into Escherichia coli and resistance to trimethoprim was measured by broth dilution and Etest. Phylogenetic analysis of previously reported dfrA genes was performed using MEGA. Long-read sequencing was conducted to determine the context of the novel dfr gene.The novel dfr gene, named dfrA34, conferred trimethoprim resistance (MIC =32?mg/L) when cloned into E. coli. Based on predicted amino acid sequences, dfrA34 shares less than 50% identity with other known dfrA genes. The dfrA34 gene is located in a class 1 integron in a multiresistance region of an IncC plasmid, adjacent to a sul gene, thus conferring clinical trimethoprim/sulfamethoxazole resistance. Additionally, dfrA34 is associated with ISCR1, enabling easy transmission between other plasmids and bacterial strains.


April 21, 2020  |  

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.


April 21, 2020  |  

Computational aspects underlying genome to phenome analysis in plants.

Recent advances in genomics technologies have greatly accelerated the progress in both fundamental plant science and applied breeding research. Concurrently, high-throughput plant phenotyping is becoming widely adopted in the plant community, promising to alleviate the phenotypic bottleneck. While these technological breakthroughs are significantly accelerating quantitative trait locus (QTL) and causal gene identification, challenges to enable even more sophisticated analyses remain. In particular, care needs to be taken to standardize, describe and conduct experiments robustly while relying on plant physiology expertise. In this article, we review the state of the art regarding genome assembly and the future potential of pangenomics in plant research. We also describe the necessity of standardizing and describing phenotypic studies using the Minimum Information About a Plant Phenotyping Experiment (MIAPPE) standard to enable the reuse and integration of phenotypic data. In addition, we show how deep phenotypic data might yield novel trait-trait correlations and review how to link phenotypic data to genomic data. Finally, we provide perspectives on the golden future of machine learning and their potential in linking phenotypes to genomic features. © 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.


April 21, 2020  |  

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.


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