Alex Bishara

Read clouds uncover variation in complex regions of the human genome

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Exploring the Tradeoffs between Programmability and Efficiency in Data-Parallel Accelerators

We present a taxonomy and modular implementation approach for data-parallel accelerators, including the MIMD, vector-SIMD, subword-SIMD, SIMT, and vector-thread (VT) architectural design patterns. We introduce Maven, a new VT microarchitecture based on the traditional vector-SIMD microarchitecture, that is considerably simpler to implement and easier to program than previous VT designs. Using an extensive design-space exploration of full VLSI implementations of many accelerator design points, we evaluate the varying tradeoffs between programmability and implementation efficiency among the MIMD, vector-SIMD, and VT patterns on a workload of compiled microbenchmarks and application kernels. We find the vector cores provide greater efficiency than the MIMD cores, even on fairly irregular kernels. Our results suggest that the Maven VT microarchitecture is superior to the traditional vector-SIMD architecture, providing both greater efficiency and easier programmability.

Human-specific NOTCH-like genes in a region linked to neurodevelopmental disorders affect cortical neurogenesis

Genetic changes causing dramatic brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and a determinant of neuronal number in the mammalian cortex. We find three paralogs of human-specific NOTCH2NL are highly expressed in radial glia cells. Functional analysis reveals different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation. NOTCH2NL genes provide the breakpoints in typical cases of 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism, and deletions with microcephaly and schizophrenia. Thus, the emergence of hominin-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger hominin neocortex accompanied by loss of genomic stability at the 1q21. 1 locus and a resulting recurrent neurodevelopmental disorder.

De novo assembly of microbial genomes from human gut metagenomes using barcoded short read sequences

Although shotgun short-read sequencing has facilitated the study of strain-level architecture within complex microbial communities, existing metagenomic approaches often cannot capture structural differences between closely related co-occurring strains. Recent methods, which employ read cloud sequencing and specialized assembly techniques, provide significantly improved genome drafts and show potential to capture these strain-level differences. Here, we apply this read cloud metagenomic approach to longitudinal stool samples from a patient undergoing hematopoietic cell transplantation. The patient9s microbiome is profoundly disrupted and is eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allows us to predict that particular mobile element integrations result in increased antibiotic resistance, which we further support using in vitro antibiotic susceptibility testing. Thus, we find read cloud sequencing to be useful in identifying strain-level differences that underlie differential fitness.Shotgun short-read sequencing methods facilitate study of the genomic content and strain-level architecture of complex microbial communities. However, existing methodologies do not capture structural differences between closely related co-occurring strains such as those arising from horizontal gene transfer and insertion sequence mobilization. Recent techniques that partition large DNA molecules, then barcode short fragments derived from them, produce short-read sequences containing long-range information. Here, we present a novel application of these short-read barcoding techniques to metagenomic samples, as well as Athena, an assembler that uses these barcodes to produce improved metagenomic assemblies. We apply our approach to longitudinal samples from the gut microbiome of a patient with a hematological malignancy. This patient underwent an intensive regimen of multiple antibiotics, chemotherapeutics and immunosuppressants, resulting in profound disruption of the microbial gut community and eventual domination by Bacteroides caccae. We significantly improve draft completeness over conventional techniques, uncover strains of B. caccae differing in the positions of transposon integration, and find the abundance of individual strains to fluctuate widely over the course of treatment. In addition, we perform RNA sequencing to investigate relative transcription of genes in B. caccae, and find overexpression of antibiotic resistance genes in our de novo assembled draft genome of B. caccae coinciding with both antibiotic administration and the appearance of proximal transposons harboring a putative bacterial promoter region. Our approach produces overall improvements in contiguity of metagenomic assembly and enables assembly of whole classes of genomic elements inaccessible to existing short-read approaches.

Culture-free generation of microbial genomes from human and marine microbiomes

Our understanding of natural microbial communities is shaped by the careful investigation of a relatively small number of isolated and cultured organisms, and by analysis of genomic sequences obtained by culture-free metagenomic sequencing approaches. Metagenomic shotgun sequencing has facilitated partial reconstruction of strain-level community structure and functional repertoire. Unfortunately, it remains difficult to cost-effectively produce high quality genome drafts for individual microbes without isolation and culture. Recent molecular techniques that partition long DNA fragments and then barcode short fragments derived from them produce “read clouds”, which are short-read sequences containing long-range information. Here, we present a novel application of a read cloud technique to microbiome samples, as well as Athena, a de novo assembler that uses these barcodes to produce improved metagenomic assemblies. We apply our approach to sequence human stool samples from two healthy individuals, and compare it to existing short read and synthetic long read metagenomic sequencing approaches. We find that read cloud metagenomic sequencing and Athena assembly produce the most complete individual genome drafts. These genome drafts are also highly contiguous (>200kb N50, <10 contigs), even for bacteria that have relatively low (20x) raw short read sequence coverage. We also apply this approach to a significantly more complex marine sediment sample and obtain 23 genome drafts with valuable 16S ribosomal RNA taxonomic marker sequences, nine of which are complete genome drafts. Read cloud metagenomic sequencing allows culture-free generation of high quality microbial genome drafts using only a single shotgun experiment.

High-quality genome sequences of uncultured microbes by assembly of read clouds

Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.

Read Clouds Uncover Variation in Complex Regions of the Human Genome

The rapid advance of next-generation sequencing (NGS) technologies has decreased the cost of genomic sequencing dramatically, enabling accurate variant discovery across whole genomes of many individuals. Current large-scale and cost-effective resequencing platforms produce reads of limited length, and as a result, reliable identification of variants within highly homologous regions of a target genome remains challenging.

The Maven vector-thread architecture

Future manycore processors will be energy-constrained, and thus the primary metric for evaluating these architectures will be their energy-efficiency. In this work, we investigate new architectural and microarchitectural mechanisms which enable a wider array of applications to be mapped to energy-efficient vector units.