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 and their data, although several times more expensive than Illumina, can be used to produce high quality genomes. Pacbio data can be used for de novo assembly, however due to its high error rate high coverage of the genome is required this raising the cost barrier. A solution for cost-effective genomes is to combine Pacbio and Illumina data leveraging the low error rates of the short Illumina reads and the length of the Pacbio reads. We have developed MaSuRCA mega-reads assembler for efficient assembly of hybrid data sets and we demonstrate that it performs well compared to the other published hybrid techniques. Another important benefit of the long reads is their ability to link the haplotype differences. The mega-reads approach corrects each Pacbio read independently and thus haplotype differences are preserved. Thus, leveraging the accuracy of the Illumina data and the length of the Pacbio reads, MaSuRCA mega-reads can produce haplotype-resolved genome assemblies, where each contig has sequence from a single haplotype. We present preliminary results on haplotype-resolved genome assemblies of faux (proof-of-concept) and real data.

April 21, 2020  |  

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.

April 21, 2020  |  

Next generation sequencing characterizes HLA diversity in a registry population from the Netherlands.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 assignments of 1009 unrelated volunteers for the unrelated donor registry in The Netherlands. The analysis characterizes all HLA exons and introns for class I alleles; at least exons 2 to 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 229 class I and 71 class II; 36 of these alleles are novel. The majority (approximately 98%) of the cumulative allele frequency at each locus is contributed by alleles that appear three or more times. Alleles encoding protein variation outside of the antigen recognition domains are 0.6% of the class I assignments and 5.3% of the class II assignments. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

April 21, 2020  |  

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test.We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs.Robust identification of CNVs by GS is possible within a clinical testing environment.

April 21, 2020  |  

Progression of the canonical reference malaria parasite genome from 2002-2019.

Here we describe the ways in which the sequence and annotation of the Plasmodium falciparum reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the P. falciparum research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of genes that were subsequently characterised experimentally and genes with significant similarity to others with known functions. In addition, the structural annotation of genes has been significantly refined; 27% of gene structures have been changed since 2002, comprising changes in exon-intron boundaries, addition or deletion of exons and the addition or deletion of genes. The sequence has also undergone significant improvements. In addition to the correction of a large number of single-base and insertion or deletion errors, a major miss-assembly between the subtelomeres of chromosome 7 and 8 has been corrected. As the number of sequenced isolates continues to grow rapidly, a single reference genome will not be an adequate basis for interpretating intra-species sequence diversity. We therefore describe in this publication a population reference genome of P. falciparum, called Pfref1. This reference will enable the community to map to regions that are not present in the current assembly. P. falciparum 3D7 will be continued to be maintained with ongoing curation ensuring continual improvements in annotation quality.

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