Several new high-quality human genome assemblies produce ethnicity-specific reference sequences and show how scientists can use this genetic information to improve precision medicine studies in Asian sub- populations. These projects demonstrate how long- read SMRT Sequencing provides robust detection of polymorphic structural variants in clinically relevant gene coding regions and phases variants into haplotypes.
In his talk from the AGBT 2015 PacBio workshop, Craig Venter detailed plans to sequence 1 million genomes and gather extensive phenotypic data to make sense of them. Included: generating 30 reference genomes to represent ethnogeographic diversity; the need for long-range continuity in sequencing; and truly predictive genomics.
Anne Deslattes Mays from Georgetown University describes how long-read sequencing provided important biological answers after three years of short-read sequencing failed to generate enough information for her gene discovery project. She says long reads are essential for understanding alternative splicing, RNA editing, and genome rearrangements.
Euan Ashley speaks about precision medicine and said clinical-grade analysis has been limited by complex regions in the human genome. His key theme,”Precision medicine needs to be accurate medicine,” was illustrated with several examples where short-read sequencing or traditional clinical sequencing methods failed to be accurate. Also included: targeted RNA sequencing and gene phasing with long-read sequencing.
Euan Ashley from Stanford University started with the premise that while current efforts in the field of genomics medicine address 30% of patient cases, there’s a need for new approaches to make sense of the remaining 70%. Toward that end, he said that accurately calling structural variants is a major need. In one translational research example, Ashley said that SMRT Sequencing with the Sequel System allowed his team to identify six potentially causative genes in an individual with complex and varied symptoms; one gene was associated with Carney syndrome, which was a match for the person’s physiology and was later…
Melissa Laird Smith discussed how the Icahn School of Medicine at Mount Sinai uses long-read sequencing for translational research. She gave several examples of targeted sequencing projects run on the Sequel System including CYP2D6, phased mutations of GLA in Fabry’s disease, structural variation breakpoint validation in glioblastoma, and full-length immune profiling of TCR sequences.
At AGBT 2017, Mike Schatz from Johns Hopkins University and Cold Spring Harbor Laboratory presented data from sequencing, assembling, and analyzing personalized, phased diploid genomes with either Illumina, 10x Genomics, and PacBio SMRT Sequencing. Compared to the short-read-based methods, PacBio data assembled in large, complete contigs and contained the broadest range of structural variants with the best resolution. Plus: unexpected translocation findings with SMRT Sequencing, validated in follow-up studies.
Personal transcriptomes in which all of an individual’s genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident…
PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions?>?50?bp. Filtering for variants that are absent in an unrelated control and overlap…
Long expansions of short tandem repeats (STRs), i.e. DNA repeats of 2-6 nt, are associated with some genetic diseases. Cost-efficient high-throughput sequencing can quickly produce billions of short reads that would be useful for uncovering disease-associated STRs. However, enumerating STRs in short reads remains largely unexplored because of the difficulty in elucidating STRs much longer than 100 bp, the typical length of short reads.We propose ab initio procedures for sensing and locating long STRs promptly by using the frequency distribution of all STRs and paired-end read information. We validated the reproducibility of this method using biological replicates and used it…
Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation…
Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only = 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger…
There is great potential for genome sequencing to enhance patient care through improved diagnostic sensitivity and more precise therapeutic targeting. To maximize this potential, genomics strategies that have been developed for genetic discovery – including DNA-sequencing technologies and analysis algorithms – need to be adapted to fit clinical needs. This will require the optimization of alignment algorithms, attention to quality-coverage metrics, tailored solutions for paralogous or low-complexity areas of the genome, and the adoption of consensus standards for variant calling and interpretation. Global sharing of this more accurate genotypic and phenotypic data will accelerate the determination of causality for novel…
The successes of targeted drugs with companion predictive biomarkers and the technological advances in gene sequencing have generated enthusiasm for evaluating personalized cancer medicine strategies using genomic profiling. We assessed the feasibility of incorporating real-time analysis of somatic mutations within exons of 19 genes into patient management. Blood, tumor biopsy and archived tumor samples were collected from 50 patients recruited from four cancer centers. Samples were analyzed using three technologies: targeted exon sequencing using Pacific Biosciences PacBio RS, multiplex somatic mutation genotyping using Sequenom MassARRAY and Sanger sequencing. An expert panel reviewed results prior to reporting to clinicians. A clinical…
In recent years, the increasing awareness that somatic mutations and other genetic aberrations drive human malignancies has led us within reach of personalized cancer medicine (PCM). The implementation of PCM is based on the following premises: genetic aberrations exist in human malignancies; a subset of these aberrations drive oncogenesis and tumor biology; these aberrations are actionable (defined as having the potential to affect management recommendations based on diagnostic, prognostic, and/or predictive implications); and there are highly specific anticancer agents available that effectively modulate these targets. This article highlights the technology underlying cancer genomics and examines the early results of genome…