June 1, 2021  |  

Progress on the reassembly and annotation of the goat genome.

The goat (Capra hircus) remains an important livestock species due to the species’ ability to forage and provide milk, meat and wool in arid environments. The current goat reference assembly and annotation borrows heavily from other loosely related livestock species, such as cattle, and may not reflect the unique structural and functional characteristics of the species. We present preliminary data from a new de novo reference assembly for goat that primarily utilizes 38 million PacBio P5-C3 reads generated from an inbred San Clemente goat. This assembly consists of only 5,902 contigs with a contig N50 size of 2.56 megabases which were grouped into scaffolds using cis-chromosome associations generated by the analysis of Hi-C sequence reads. To provide accurate functional genetic annotation, we utilized existing RNA-seq data and generated new data consisting of over 784 million reads from a combination of 27 different developmental timepoints/tissues. This dataset provides a tangible improvement over existing goat genomics resources by correcting over 247 misassemblies in the current goat reference genome and by annotating predicted gene models with actual expressed transcript data. Our goal is to provide a high quality resource to researchers to enable future genomic selection and functional prediction within the field of goat genomics.


June 1, 2021  |  

A genome assembly of the domestic goat from 70x coverage of single molecule, real-time sequence.

Goat is an important source of milk, meat, and fiber, especially in developing countries. An advantage of goats as livestock is the low maintenance requirements and high adaptability compared to other milk producers. The global population of domestic goats exceeds 800 million. In Africa, goat production is characterized by low productivity levels, and attempts to introduce more productive breeds have met with poor success due in part to nutritional constraints. It has been suggested that incorporation of selective breeding within the herds adapted for survival could represent one approach to improving food security across Africa. A recently produced genome assembly of a Chinese Yunnan breed goat, based on 192 Gb of short reads across a range of insert sizes from 180 bp to 20 kb, reported a contig N50 of 18.7 kb. The scaffold N50 was improved from 2.2 Mb to 3.1 Mb by addition of fosmid end sequence, with an estimated 140 million Ns in gaps and 91% coverage. The assembly has proven somewhat problematic for pursuing genome-wide association analysis with SNP arrays, apparently due in part to errors in ordering of markers using the draft genome. In order to provide a higher quality assembly, we sequenced a highly inbred, San Clemente breed goat genome using 458 SMRT cells on the Pacific Biosciences platform. These cells generated 193.5 Gbases of sequence after processing into subreads, with mean 5110 bases and max subread length of 40.5 kb. This sequence data generated an assembly using the recently reported MHAP error correction approach and Celera Assembler v8.2. The contig N50 was 2.5 Mb, with the largest contig spanning 19.5 Mb. Additional characteristics of the assembly will be presented.


June 1, 2021  |  

Toward comprehensive genomics analysis with de novo assembly.

Whole genome sequencing can provide comprehensive information important for determining the biochemical and genetic nature of all elements inside a genome. The high-quality genome references produced from past genome projects and advances in short-read sequencing technologies have enabled quick and cheap analysis for simple variants. However even with the focus on genome-wide resequencing for SNPs, the heritability of more than 50% of human diseases remains elusive. For non-human organisms, high-contiguity references are deficient, limiting the analysis of genomic features. The long and unbiased reads from single molecule, real-time (SMRT) Sequencing and new de novo assembly approaches have demonstrated the ability to detect more complicated variants and chromosome-level phasing. Moreover, with the recent advance of bioinformatics algorithms and tools, the computation tasks for completing high-quality de novo assembly of large genomes becomes feasible with commodity hardware. Ongoing development in sequencing technologies and bioinformatics will likely lead to routine generation of high-quality reference assemblies in the future. We discuss the current state of art and the challenges in bioinformatics toward such a goal. More specifically, explicit examples of pragmatic computational requirements for assembling mammalian-size genomes and algorithms suitable for processing diploid genomes are discussed.


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.


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  |  

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.


June 1, 2021  |  

From Sequencing to Chromosomes: New de novo assembly and scaffolding methods improve the goat reference genome

Single-molecule sequencing is now routinely used to assemble complete, high-quality microbial genomes, but these assembly methods have not scaled well to large genomes. To address this problem, we previously introduced the MinHash Alignment Process (MHAP) for overlapping single-molecule reads using probabilistic, locality-sensitive hashing. Integrating MHAP with Celera Assembler (CA) has enabled reference-grade assemblies of model organisms, revealing novel heterochromatic sequences and filling low-complexity gap sequences in the GRCh38 human reference genome. We have applied our methods to assemble the San Clemente goat genome. Combining single-molecule sequencing from Pacific Biosciences and BioNano Genomics generates and assembly that is over 150-fold more contiguous than the latest Capra hircus reference. In combination with Hi-C sequencing, the assembly surpasses reference assemblies, de novo, with minimal manual intervention. The autosomes are each assembled into a single scaffold. Our assembly provides a more complete gene reconstruction, better alignments with Goat 52k chip, and improved allosome reconstruction. In addition to providing increased continuity of sequence, our assembly achieves a higher BUSCO completion score (84%) than the existing goat reference assembly suggesting better quality annotation of gene models. Our results demonstrate that single-molecule sequencing can produce near-complete eukaryotic genomes at modest cost and minimal manual effort.


June 1, 2021  |  

Progress Toward a Low Budget Reference Grade Genome Assembly

Reference quality de novo genome assemblies were once solely the domain of large, well-funded genome projects. While next-generation short read technology removed some of the cost barriers, accurate chromosome-scale assembly remains a real challenge. Here we present efforts to de novo assemble the goat (Capra hircus) genome. Through the combination of single-molecule technologies from Pacific Biosciences (sequencing) and BioNano Genomics (optical mapping) coupled with high-throughput chromosome conformation capture sequencing (Hi-C), an inbred San Clemente goat genome has been sequenced and assembled to a high degree of completeness at a relatively modest cost. Starting with 38 million PacBio reads, we integrated the MinHash Alignment Process (MHAP) with the Celera Assembler (CA) to produce an assembly composed of 3110 contigs with a contig N50 size of 4.7 Mb. This assembly was scaffolded with BioNano genome maps derived from a single IrysChip into 333 scaffolds with an N50 of 23.1 Mb including the complete scaffolding of chromosome 20. Finally, cis-chromosome associations were determined by Hi-C, yielding complete reconstruction of all autosomes into single scaffolds with a final N50 of 91.7 Mb. We hope to demonstrate that our methods are not only cost effective, but improve our ability to annotate challenging genomic regions such as highly repetitive immune gene clusters.


June 1, 2021  |  

How to Compare and Cluster Every Known Genome in about an Hour

Given a massive collection of sequences, it is infeasible to perform pairwise alignment for basic tasks like sequence clustering and search. To address this problem, we demonstrate that the MinHash technique, first applied to clustering web pages, can be applied to biological sequences with similar effect, and extend this idea to include biologically relevant distance and significance measures. Our new tool, Mash, uses MinHash locality-sensitive hashing to reduce large sequences to a representative sketch and rapidly estimate pairwise distances between genomes or metagenomes. Using Mash, we explored several use cases, including a 5,000-fold size reduction and clustering of all 55,000 NCBI RefSeq genomes in 46 CPU hours. The resulting 93 MB sketch database includes all RefSeq genomes, effectively delineates known species boundaries, reconstructs approximate phylogenies, and can be searched in seconds using assembled genomes or raw sequencing runs from Illumina, Pacific Biosciences, and Oxford Nanopore. For metagenomics, Mash scales to thousands of samples and can replicate Human Microbiome Project and Global Ocean Survey results in a fraction of the time. Other potential applications include any problem where an approximate, global sequence distance is acceptable, e.g. to triage and cluster sequence data, assign species labels to unknown genomes, quickly identify mis- tracked samples, and search massive genomic databases. In addition, the Mash distance metric is based on simple set intersections, which are compatible with homomorphic encryption schemes. To facilitate integration with other software, Mash is implemented as a lightweight C++ toolkit and freely released under a BSD license athttps://github.com/marbl/mash


June 1, 2021  |  

A high-quality genome assembly of SMRT Sequences reveals long-range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, chikungunya, and other diseases. The outbreak of Zika in the Americas, which can cause microcephaly in the fetus of infected women, adds urgency to the need for a high-quality reference genome in order to better understand the organism’s biology and its role in transmitting human disease. We describe the first diploid assembly of an insect genome, using SMRT sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.


June 1, 2021  |  

A high-quality genome assembly of SMRT sequences reveals long range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, and chikungunya. We describe the first diploid assembly of an insect genome, using SMRT Sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30 after polishing). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence with dramatic differences in coding sequence content, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.


June 1, 2021  |  

FALCON-Phase integrates PacBio and HiC data for de novo assembly, scaffolding and phasing of a diploid Puerto Rican genome (HG00733)

Haplotype-resolved genomes are important for understanding how combinations of variants impact phenotypes. The study of disease, quantitative traits, forensics, and organ donor matching are aided by phased genomes. Phase is commonly resolved using familial data, population-based imputation, or by isolating and sequencing single haplotypes using fosmids, BACs, or haploid tissues. Because these methods can be prohibitively expensive, or samples may not be available, alternative approaches are required. de novo genome assembly with PacBio Single Molecule, Real-Time (SMRT) data produces highly contiguous, accurate assemblies. For non-inbred samples, including humans, the separate resolution of haplotypes results in higher base accuracy and more contiguous assembled sequences. Two primary methods exist for phased diploid genome assembly. The first, TrioCanu requires Illumina data from parents and PacBio data from the offspring. The long reads from the child are partitioned into maternal and paternal bins using parent-specific sequences; the separate PacBio read bins are then assembled, generating two fully phased genomes. An alternative approach (FALCON-Unzip) does not require parental information and separates PacBio reads, during genome assembly, using heterozygous SNPs. The length of haplotype phase blocks in FALCON-Unzip is limited by the magnitude and distribution of heterozygosity, the length of sequence reads, and read coverage. Because of this, FALCON-Unzip contigs typically contain haplotype-switch errors between phase blocks, resulting in primary contig of mixed parental origin. We developed FALCON-Phase, which integrates Hi-C data downstream of FALCON-Unzip to resolve phase switches along contigs. We applied the method to a human (Puerto Rican, HG00733) and non-human genome assemblies and evaluated accuracy using samples with trio data. In a cattle genome, we observe >96% accuracy in phasing when compared to TrioCanu assemblies as well as parental SNPs. For a high-quality PacBio assembly (>90-fold Sequel coverage) of a Puerto Rican individual we scaffolded the FALCON-Phase contigs, and re-phased the contigs creating a de novo scaffolded, phased diploid assembly with chromosome-scale contiguity.


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.


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