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Auto Tag: N50

June 1, 2021  |  

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine.

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environments.

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June 1, 2021  |  

MinHash for overlapping and assembly

2015 SMRT Informatics Developers Conference Presentation Slides: Sergey Koren of National Biodefense Analysis and Countermeasures Center (NBACC) provided an overview of the MHAP algorithm, a method for assembling large genomes with Sing-Molecule Sequencing and locality sensitive hashing. Using MHAP, Koren produced a human assembly (CHM1) with a contig N50 of >23 Mb.

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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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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 is adapting these methods and developing new methods to characterize 2 families, one Asian and one Ashkenazi Jewish from the Personal Genome Project, which are consented for public release of sequencing and phenotype data. We have generated a larger and even more diverse data set on these samples, including high-depth Illumina paired-end and mate-pair, Complete Genomics, and Ion Torrent short-read data, as well as Moleculo, 10X, Oxford Nanopore, PacBio, and BioNano Genomics long-read data. We are analyzing these data to provide an accurate assessment of not just small variants but also large structural variants (SVs) in both “easy” regions of the genome and in some “hard” repetitive regions. We have also made all of the input data sources publicly available for download, analysis, and publication.Results: Our arbitration method produced a reference data set of 2,787,291 single nucleotide variants (SNVs), 365,135 indels, 2744 SVs, and 2.2 billion homozygous reference calls for our pilot genome. We found that our call set is highly sensitive and specific in comparison to independent reference data sets. We have also generated preliminary assemblies and structural variant calls for the next 2 trios from long read data and are currently integrating and validating these.Discussion: We combined the strengths of each of our input datasets to develop a comprehensive and accurate benchmark call set. In the short time it has been available, over 20 published or submitted papers have used our data. Many challenges exist in comparing to our benchmark calls, and thus we have worked with the Global Alliance for Genomics and Health to develop standardized methods, performance metrics, and software to assist in its use.[1] Zook et al, Nat Biotech. 2014.

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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), which are essentially haploid. CHM13 was sequenced with the latest PacBio technology (P6-C5) to 52X genome coverage and assembled using Daligner and Falcon v0.2 (GCA_000983455.1, CHM13_1.1). Compared to the first mole (CHM1) PacBio assembly (GCA_001007805.1, 54X) contig N50 of 4.5Mb, the contig N50 of CHM13_1.1 is almost 13Mb, and there is a 13-fold reduction in the number of contigs. This demonstrates the improved contiguity of sequence generated with the new chemistry. We annotated 50,188 RefSeq transcripts of which only 0.63% were split transcripts, and the repetitive and segmental duplication content was within the expected range. These data all indicate an extremely high quality assembly. Additionally, we sequenced CHM13 DNA using Illumina SBS technology to 60X coverage, aligned these reads to the GRCh37, GRCh38, and CHM13_1.1 assemblies and performed variant calling using the SpeedSeq pipeline. The number of single nucleotide variants (SNV) and indels was comparable between GRCh37 and GRCh38. Regions that showed increased SNV density in GRCh38 compared to GRCh37 could be attributed to the addition of centromeric alpha satellite sequence to the reference assembly. Alternatively, regions of decreased SNV density in GRCh38 were concentrated in regions that were improved from BAC based sequencing of CHM1 such as 1p12 and 1q21 containing the SRGAP2 gene family. The alignment of PacBio reads to GRCh37 and GRCh38 assemblies allowed us to resolve complex loci such as the MHC region where the best alignment was to the DBB (A2-B57-DR7) haplotype. Finally, we will discuss how combining the two high quality mole assemblies can be used for benchmarking and novel bioinformatics tool development.

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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 and other important mutations may be missed altogether because of repeats, multi-mapping reads, and the failure to reliably anchor alignments to both sides of a variation. To address these challenges, we have sequenced SK-BR-3 using PacBio long read technology. Using the new P6-C4 chemistry, we generated more than 70X coverage of the genome with average read lengths of 9-13kb (max: 71kb). Using Lumpy for split-read alignment analysis, as well as our novel assembly-based algorithms for finding complex variants, we have developed a detailed map of structural variations in this cell line. Taking advantage of the newly identified breakpoints and combining these with copy number assignments, we have developed an algorithm to reconstruct the mutational history of this cancer genome. From this we have discovered a complex series of nested duplications and translocations between chr17 and chr8, two of the most frequent translocation partners in primary breast cancers, resulting in amplification of HER2. We have also carried out full-length transcriptome sequencing using PacBio’s Iso-Seq technology, which has revealed a number of previously unrecognized gene fusions and isoforms. Combining long-read genome and transcriptome sequencing technologies enables an in-depth analysis of how changes in the genome affect the transcriptome, including how gene fusions are created across multiple chromosomes. This analysis has established the most complete cancer reference genome available to date, and is already opening the door to applying long-read sequencing to patient samples with complex genome structures.

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June 1, 2021  |  

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of Macrostomum lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130X coverage by long sequencing reads from the PacBio platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene expression patterns during regeneration, examining pathways important to stem cell function. As a whole, our data will provide a crucial resource for the community for the study not only of invertebrate evolution and phylogeny but also of regeneration and somatic pluripotency.

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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 SMRT Technology, which will allow detailed analysis of genes associated with immunity, behavior, and learning. Using SMRT Sequencing, we present here best practices for achieving long reads for whole genome shotgun sequencing for complex plant and animal genomes such as the ‘alala genome. With recent advances in SMRTbell library preparation, P6-C4 chemistry and 6-hour movies, the number of useable bases now exceeds 1 Gb per SMRT Cell. Read lengths averaging 10 – 15 kb can be routinely achieved, with the longest reads approaching 70 kb. Furthermore, > 25% of useable bases are in reads greater than 30 kb, advantageous for generating contiguous draft assemblies of contig N50 up to 5 Mb. De novo assemblies of large genomes are now more tractable using SMRT Sequencing as the standalone technology. We also present guidelines for planning out projects for the de novo assembly of large genomes.

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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 purifying selection, suggesting they are beneficial to the host. To characterize EVE biogenesis in a tractable system, we sequenced the Ae. aegypti cell line, Aag2, to 58-fold coverage and present a de novo assembly of the genome. The assembly contains 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, consisting of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify EVEs in the genome homologous to a range of extant viruses, many of which cluster in these regions of repetitive DNA. The contiguous assembly allows for more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing.

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June 1, 2021  |  

An update on goat genomics

Goats are specialized in dairy, meat and fiber production, being adapted to a wide range of environmental conditions and having a large economic impact in developing countries. In the last years, there have been dramatic advances in the knowledge of the structure and diversity of the goat genome/transcriptome and in the development of genomic tools, rapidly narrowing the gap between goat and related species such as cattle and sheep. Major advances are: 1) publication of a de novo goat genome reference sequence; 2) Development of whole genome high density RH maps, and; 3) Design of a commercial 50K SNP array. Moreover, there are currently several projects aiming at improving current genomic tools and resources. An improved assembly of the goat genome using PacBio reads is being produced, and the design of new SNP arrays is being studied to accommodate the specific needs of this species in the context of very large scale genotyping projects (i.e. breed characterization at an international scale and genomic selection) and parentage analysis. As in other species, the focus has now turned to the identification of causative mutations underlying the phenotypic variation of traits. In addition, since 2014, the ADAPTmap project (www.goatadaptmap.org) has gathered data to explore the diversity of caprine populations at a worldwide scale by using a wide variety of approaches and data.

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June 1, 2021  |  

Improving the goat long-read assembly with optical mapping and Hi-C scaffolding

Reference genome assemblies provide important context in genetics by standardizing the order of genes and providing a universal set of coordinates for individual nucleotides. Often due to the high complexity of genic regions and higher copy number of genes involved in immune function, immunity-related genes are often misassembled in current reference assemblies. This problem is particularly ubiquitous in the reference genomes of non-model organisms as they often do not receive the years of curation necessary to resolve annotation and assembly errors. In this study, we reassemble a reference genome of the goat (Capra hircus) using modern PacBio technology in tandem with BioNano Genomics Irys optical maps and Lachesis clustering in order to provide a high quality reference assembly without the need for extensive filtering. Initial PacBio assemblies using P5C4 chemistry achieved contig N50’s of 4 Megabases and a BUSCO completion score of 84.0%, which is comparable to several finished model organism reference assemblies. We used BioNano Genomics’ Irys platform to generate 336 scaffolds from this data with a scaffold N50 of 24 megabases and total genome coverage of 98%. Lachesis interaction maps were used with a clustering algorithm to associate Irys scaffolds into the expected 30 chromosome physical maps. Comparisons of the initial hybrid scaffolds generated from the long read contigs and optical map information to a previously generated RH map revealed that the entirety of the Goat autosome 20 physical map was contained within one scaffold. Additionally, the BioNano scaffolding resolved several difficult regions that contained genes related to innate immunity which were problem regions in previous reference genome assemblies.

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June 1, 2021  |  

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46 SMRT cells) reads, 33,720 unigenes were de novo assembled. Comparison of sequencing technologies revealed improved coverage with Illumina HiSeq reads and better splice variant detection with PacBio Iso-Seq reads. The genes identified as unique to each library ranges from 199 transcripts (basket seedling) to 3,482 transcripts (female cones). In total, 10,026 transcripts were shared by all libraries. Genes differentially expressed in response to these provided insight on abiotic and biotic stress responses. To analyze orthologous sequences, we compared the translated sequences against 19 plant species, identifying 7,229 transcripts that clustered uniquely among the conifers. We have generated here a high quality transcriptome from one WPBR susceptible and one WPBR resistant sugar pine individual. Through the comprehensive tissue sampling and the depth of the sequencing achieved, detailed information on disease resistance can be further examined.

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