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Tuesday, 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.

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Tuesday, June 1, 2021

Genome in a Bottle: You’ve sequenced. How well did you do?

Purpose: Clinical laboratories, research laboratories and technology developers all need DNA samples with reliably known genotypes in order to help validate and improve their methods. The Genome in a Bottle Consortium (genomeinabottle.org) has been developing Reference Materials with high-accuracy whole genome sequences to support these efforts.Methodology: Our pilot reference material is based on Coriell sample NA12878 and was released in May 2015 as NIST RM 8398 (tinyurl.com/giabpilot). To minimize bias and improve accuracy, 11 whole-genome and 3 exome data sets produced using 5 different technologies were integrated using a systematic arbitration method [1]. The Genome in a Bottle Analysis Group…

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Tuesday, June 1, 2021

Building a platinum human genome assembly from single haplotype human genomes generated from long molecule sequencing

The human reference sequence has provided a foundation for studies of genome structure, human variation, evolutionary biology, and disease. At the time the reference was originally completed there were some loci recalcitrant to closure; however, the degree to which structural variation and diversity affected our ability to produce a representative genome sequence at these loci was still unknown. Many of these regions in the genome are associated with large, repetitive sequences and exhibit complex allelic diversity such producing a single, haploid representation is not possible. To overcome this challenge, we have sequenced DNA from two hydatidiform moles (CHM1 and CHM13),…

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Tuesday, June 1, 2021

Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing

Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene. However, these technologies cannot precisely characterize the nature and context of the identified genomic events…

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Tuesday, June 1, 2021

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by…

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Tuesday, June 1, 2021

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under…

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Tuesday, June 1, 2021

MaSuRCA Mega-Reads Assembly Technique for haplotype resolved genome assembly of hybrid PacBio and Illumina Data

The developments in DNA sequencing technology over the past several years have enabled large number of scientists to obtain sequences for the genomes of their interest at a fairly low cost. Illumina Sequencing was the dominant whole genome sequencing technology over the past few years due to its low cost. The Illumina reads are short (up to 300bp) and thus most of those draft genomes produced from Illumina data are very fragmented which limits their usability in practical scenarios. Longer reads are needed for more contiguous genomes. Recently Pacbio sequencing made significant advances in developing cost-effective long-read (>10000bp) sequencing technology…

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Tuesday, June 1, 2021

Diploid genome assembly and comprehensive haplotype sequence reconstruction

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 generate two datasets for developing the algorithms and the prototype software:…

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Tuesday, 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…

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Tuesday, June 1, 2021

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue and Zika viruses by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are…

Read More »

Tuesday, June 1, 2021

Immune regions are no longer incomprehensible with SMRT Sequencing

The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these…

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Tuesday, June 1, 2021

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are both important in understanding the genetic basis for human disease, and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid-aware de novo assembly of Craig Venter’s well-studied genome.

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