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.


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.


June 1, 2021  |  

Structural variant combining Illumina and low-coverage PacBio

Structural variant calling combining Illumina and low-coverage Pacbio Detection of large genomic variation (structural variants) has proven challenging using short-read methods. Long-read approaches which can span these large events have promise to dramatically expand the ability to accurately call structural variants. Although sequencing with Pacific Biosciences (Pacbio) long-read technology has become increasingly high throughput, generating high coverage with the technology can still be limiting and investigators often would like to know what pacbio coverages are adequate to call structural variants. Here, we present a method to identify a substantially higher fraction of structural variants in the human genome using low-coverage pacbio data by multiple strategies for ensembling data types and algorithms. Algorithmically, we combine three structural variant callers: PBHoney by Adam English, Sniffles by Fritz Sedlazeck, and Parliament by Adam English (which we have modified to improve for speed). Parliament itself uses a combination of Pacbio and Illumina data with a number of short-read callers (Breakdancer, Pindel, Crest, CNVnator, Delly, and Lumpy). We show that the outputs of these three programs are largely complementary to each other, with each able to uniquely access different sets of structural variants at different coverages. Combining them together can more than double the recall of true structural variants from a truth set relative to sequencing with Illumina alone, with substantial improvements even at low pacbio coverages (3x – 7x). This allows us to present for the first time cost-benefit tradeoffs to investigators about how much pacbio sequencing will yield what improvements in SV-calling. This work also builds upon the foundational work of Genome in a Bottle led by Justin Zook in establishing a truth set for structural variants in the Ashkenazim-Jewish trio data recently released. This work demonstrates the power of this benchmark set – one of the first of its kind for structural variation data – to help understand and refine the accuracies of calling structural variants with a number of approaches.


June 1, 2021  |  

Effect of coverage depth and haplotype phasing on structural variant detection with PacBio long reads

Each human genome has thousands of structural variants compared to the reference assembly, up to 85% of which are difficult or impossible to detect with Illumina short reads and are only visible with long, multi-kilobase reads. The PacBio RS II and Sequel single molecule, real-time (SMRT) sequencing platforms have made it practical to generate long reads at high throughput. These platforms enable the discovery of structural variants just as short-read platforms did for single nucleotide variants. Numerous software algorithms call structural variants effectively from PacBio long reads, but algorithm sensitivity is lower for insertion variants and all heterozygous variants. Furthermore, the impact of coverage depth and read lengths on sensitivity is not fully characterized. To quantify how zygosity, coverage depth, and read lengths impact the sensitivity of structural variant detection, we obtained high coverage PacBio sequences for three human samples: haploid CHM1, diploid NA12878, and diploid SK-BR-3. For each dataset, reads were randomly subsampled to titrate coverage from 0.5- to 50-fold. The structural variants detected at each coverage were compared to the set at “full” 50-fold coverage. For the diploid samples, additional titrations were performed with reads first partitioned by phase using single nucleotide variants for essentially haploid structural variant discovery. Even at low coverages (1- to 5-fold), PacBio long reads reveal hundreds of structural variants that are not seen in deep 50-fold Illumina whole genome sequences. At moderate 10-fold PacBio coverage, a majority of structural variants are detected. Sensitivity begins to level off at around 40-fold coverage, though it does not fully saturate before 50-fold. Phasing improves sensitivity for all variant types, especially at moderate 10- to 20-fold coverage. Long reads are an effective tool to identify and phase structural variants in the human genome. The majority of variants are detected at moderate 10-fold coverage, and even extremely low long-read coverage (1- to 5-fold) reveals variants that are invisible to short-read sequencing. Performance will continue to improve with better software and longer reads, which will empower studies to connect structural variants to healthy and disease traits in the human population.


June 1, 2021  |  

Single molecule high-fidelity (HiFi) Sequencing with >10 kb libraries

Recent improvements in sequencing chemistry and instrument performance combine to create a new PacBio data type, Single Molecule High-Fidelity reads (HiFi reads). Increased read length and improvement in library construction enables average read lengths of 10-20 kb with average sequence identity greater than 99% from raw single molecule reads. The resulting reads have the accuracy comparable to short read NGS but with 50-100 times longer read length. Here we benchmark the performance of this data type by sequencing and genotyping the Genome in a Bottle (GIAB) HG0002 human reference sample from the National Institute of Standards and Technology (NIST). We further demonstrate the general utility of HiFi reads by analyzing multiple clones of Cabernet Sauvignon. Three different clones were sequenced and de novo assembled with the CANU assembly algorithm, generating draft assemblies of very high contiguity equal to or better than earlier assembly efforts using PacBio long reads. Using the Cabernet Sauvignon Clone 8 assembly as a reference, we mapped the HiFi reads generated from Clone 6 and Clone 47 to identify single nucleotide polymorphisms (SNPs) and structural variants (SVs) that are specific to each of the three samples.


April 21, 2020  |  

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes. © 2019 John Wiley & Sons Ltd/University College London.


April 21, 2020  |  

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

As they migrated out of Africa and into Europe and Asia, anatomically modern humans interbred with archaic hominins, such as Neanderthals and Denisovans. The result of this genetic introgression on the recipient populations has been of considerable interest, especially in cases of selection for specific archaic genetic variants. Hsieh et al. characterized adaptive structural variants and copy number variants that are likely targets of positive selection in Melanesians. Focusing on population-specific regions of the genome that carry duplicated genes and show an excess of amino acid replacements provides evidence for one of the mechanisms by which genetic novelty can arise and result in differentiation between human genomes.Science, this issue p. eaax2083INTRODUCTIONCharacterizing genetic variants underlying local adaptations in human populations is one of the central goals of evolutionary research. Most studies have focused on adaptive single-nucleotide variants that either arose as new beneficial mutations or were introduced after interbreeding with our now-extinct relatives, including Neanderthals and Denisovans. The adaptive role of copy number variants (CNVs), another well-known form of genomic variation generated through deletions or duplications that affect more base pairs in the genome, is less well understood, despite evidence that such mutations are subject to stronger selective pressures.RATIONALEThis study focuses on the discovery of introgressed and adaptive CNVs that have become enriched in specific human populations. We combine whole-genome CNV calling and population genetic inference methods to discover CNVs and then assess signals of selection after controlling for demographic history. We examine 266 publicly available modern human genomes from the Simons Genome Diversity Project and genomes of three ancient homininstextemdasha Denisovan, a Neanderthal from the Altai Mountains in Siberia, and a Neanderthal from Croatia. We apply long-read sequencing methods to sequence-resolve complex CNVs of interest specifically in the Melanesianstextemdashan Oceanian population distributed from Papua New Guinea to as far east as the islands of Fiji and known to harbor some of the greatest amounts of Neanderthal and Denisovan ancestry.RESULTSConsistent with the hypothesis of archaic introgression outside Africa, we find a significant excess of CNV sharing between modern non-African populations and archaic hominins (P = 0.039). Among Melanesians, we observe an enrichment of CNVs with potential signals of positive selection (n = 37 CNVs), of which 19 CNVs likely introgressed from archaic hominins. We show that Melanesian-stratified CNVs are significantly associated with signals of positive selection (P = 0.0323). Many map near or within genes associated with metabolism (e.g., ACOT1 and ACOT2), development and cell cycle or signaling (e.g., TNFRSF10D and CDK11A and CDK11B), or immune response (e.g., IFNLR1). We characterize two of the largest and most complex CNVs on chromosomes 16p11.2 and 8p21.3 that introgressed from Denisovans and Neanderthals, respectively, and are absent from most other human populations. At chromosome 16p11.2, we sequence-resolve a large duplication of >383 thousand base pairs (kbp) that originated from Denisovans and introgressed into the ancestral Melanesian population 60,000 to 170,000 years ago. This large duplication occurs at high frequency (>79%) in diverse Melanesian groups, shows signatures of positive selection, and maps adjacent to Homo sapienstextendashspecific duplications that predispose to rearrangements associated with autism. On chromosome 8p21.3, we identify a Melanesian haplotype that carries two CNVs, a ~6-kbp deletion, and a ~38-kbp duplication, with a Neanderthal origin and that introgressed into non-Africans 40,000 to 120,000 years ago. This CNV haplotype occurs at high frequency (44%) and shows signals consistent with a partial selective sweep in Melanesians. Using long-read sequencing genomic and transcriptomic data, we reconstruct the structure and complex evolutionary history for these two CNVs and discover previously undescribed duplicated genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that show an excess of amino acid replacements consistent with the action of positive selection.CONCLUSIONOur results suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation that is absent from current reference genomes.Large adaptive-introgressed CNVs at chromosomes 8p21.3 and 16p11.2 in Melanesians.The magnifying glasses highlight structural differences between the archaic (top) and reference (bottom) genomes. Neanderthal (red) and Denisovan (blue) haplotypes encompassing large CNVs occur at high frequencies in Melanesians (44 and 79%, respectively) but are absent (black) in all non-Melanesians. These CNVs create positively selected genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that are absent from the reference genome.Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.


April 21, 2020  |  

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.


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