Sara Goodwin

Coming of age: ten years of next-generation sequencing technologies

Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between ∼5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

Oxford Nanopore Sequencing and de novo Assembly of a Eukaryotic Genome

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available that we used for sequencing the S. cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr (https://github.com/jgurtowski/nanocorr) specifically for Oxford Nanopore reads, as existing packages were incapable of assembling the long read lengths (5-50kbp) at such high error rate (between ∼5 and 40% error). With this new method we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: the contig N50 length is more than ten-times greater than an Illumina-only assembly (678kb versus 59.9kbp), and has greater than 99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly. Reviewer link to data http://schatzlab.cshl.edu/data/nanocorr/

Third-generation sequencing and the future of genomics

Third-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address longstanding problems in de novo genome assembly, structural variation analysis and haplotype phasing.

Next-Generation Sequencing as Input for Chemometrics in Differential Sensing Routines

Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi-specific receptors, Next-Gen DNA sequencing was used to generate data, and cell line differentiation was the test-bed application. The study of a principal component analysis loading plot revealed cross-reactivity between the aptamers. The technique generates high-dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid-based receptors exist.

A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing

Maize and sorghum are both important crops with similar overall plant architectures, but they have key differences, especially in regard to their inflorescences. To better understand these two organisms at the molecular level, we compared expression profiles of both protein-coding and noncoding transcripts in 11 matched tissues using single-molecule, long-read, deep RNA sequencing. This comparative analysis revealed large numbers of novel isoforms in both species. Evolutionarily young genes were likely to be generated in reproductive tissues and usually had fewer isoforms than old genes. We also observed similarities and differences in alternative splicing patterns and activities, both among tissues and between species. The maize subgenomes exhibited no bias in isoform generation; however, genes in the B genome were more highly expressed in pollen tissue, whereas genes in the A genome were more highly expressed in endosperm. We also identified a number of splicing events conserved between maize and sorghum. In addition, we generated comprehensive and high-resolution maps of poly(A) sites, revealing similarities and differences in mRNA cleavage between the two species. Overall, our results reveal considerable splicing and expression diversity between sorghum and maize, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two species.

1D Genome Sequencing on the Oxford Nanopore MinION

Todays short‐read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high‐throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become indispensable tools for many researchers. With greater insight has come the revelation that many genomes are much more complicated than originally thought and include many rearrangements and copy‐number variations. Unfortunately, short‐read sequencing technologies are not well suited for identifying many of these types of events. Long‐read sequencing technologies can read contiguous fragments of DNA in excess of 10 kb and are much better suited for detecting large structural events. The newest long‐read sequencing instrument is the MinION device from Oxford Nanopore. The rapid sequencing speed and low upfront instrument cost are features drawing interest in this device from the genomics community. This unit provides a representative protocol for carrying out human genome sequencing on the Oxford Nanopore MinION.

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.

SiLiCO: A Simulator of Long Read Sequencing in PacBio and Oxford Nanopore

Summary Long read sequencing platforms, which include the widely used Pacific Biosciences (PacBio) platform and the emerging Oxford Nanopore platform, aim to produce sequence fragments in excess of 15-20 kilobases, and have proved advantageous in the identification of structural variants and easing genome assembly. However, long read sequencing remains relatively expensive and error prone, and failed sequencing runs represent a significant problem for genomics core facilities. To quantitatively assess the underlying mechanics of sequencing failure, it is essential to have highly reproducible and controllable reference data sets to which sequencing results can be compared. Here, we present SiLiCO, the first in silico simulation tool to generate standardized sequencing results from both of the leading long read sequencing platforms. Availability SiLiCO is an open source package written in Python. It is freely available at https://www.github.com/ethanagbaker/SiLiCO under the GNU GPL 3.0 license. Contact Supplementary information Supplementary data are available at Bioinformatics online.