Museomics of a rare taxon: placing Whalleyanidae in the Lepidoptera Tree of Life

AbstractMuseomics is a valuable tool that utilises the diverse biobanks that are natural history museums. The ability to sequence genomes from old specimens has expanded not only the variety of interesting taxa available to study but also the scope of questions that can be investigated in order to further knowledge about biodiversity. Here we present whole genome sequencing results from the enigmatic genus Whalleyana, as well as the families Callidulidae and Hyblaeidae. Library preparation was carried out on four museum specimens and one existing DNA extract and sequenced with Illumina short reads. De novo assembly resulted in highly fragmented genomes with the N50 ranging from 317 – 2,078 bp. Mining of a manually curated gene set of 332 genes from these draft genomes had an overall gene recovery rate of 64 – 90%. Phylogenetic analysis places Whalleyana as sister to Callidulidae, and Hyblaea as sister to Pyraloidea. Since the former sister-group relationship turns out to be also supported by ten morphological synapomorphies, we propose to formally assign the Whalleyanidae to the superfamily Calliduloidea. These results highlight the usefulness of not only museum specimens, but also existing DNA extracts, for whole genome sequencing and gene mining for phylogenomic studies.

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Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly

BMC Research Notes ◽

10.1186/s13104-016-2072-9 ◽

2016 ◽

Vol 9 (1) ◽

Cited By ~ 37

Author(s):

Christopher Huptas ◽

Siegfried Scherer ◽

Mareike Wenning

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

De Novo Assembly ◽

De Novo ◽

Whole Genome ◽

Library Preparation ◽

Factors Influencing

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Effective variant filtering and expected candidate variant yield in studies of rare human disease

npj Genomic Medicine ◽

10.1038/s41525-021-00227-3 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Brent S. Pedersen ◽

Joe M. Brown ◽

Harriet Dashnow ◽

Amelia D. Wallace ◽

Matt Velinder ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Rare Disease ◽

Genome Sequencing ◽

Autosomal Dominant ◽

De Novo ◽

Autosomal Dominant Inheritance ◽

Compound Heterozygous ◽

Whole Genome ◽

Dominant Inheritance ◽

Family Based

AbstractIn studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

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A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Scientific Reports ◽

10.1038/s41598-020-80031-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kathy E. Raven ◽

Sophia T. Girgis ◽

Asha Akram ◽

Beth Blane ◽

Danielle Leek ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Clinical Microbiology ◽

Bacterial Species ◽

Outbreak Detection ◽

Whole Genome ◽

Library Preparation ◽

Simultaneous Processing ◽

Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

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Norgal: extraction and de novo assembly of mitochondrial DNA from whole-genome sequencing data

BMC Bioinformatics ◽

10.1186/s12859-017-1927-y ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 21

Author(s):

Kosai Al-Nakeeb ◽

Thomas Nordahl Petersen ◽

Thomas Sicheritz-Pontén

Keyword(s):

Mitochondrial Dna ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

De Novo Assembly ◽

De Novo ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

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Evaluating coverage bias in next-generation sequencing of Escherichia coli

PLoS ONE ◽

10.1371/journal.pone.0253440 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0253440

Author(s):

Samantha Gunasekera ◽

Sam Abraham ◽

Marc Stegger ◽

Stanley Pang ◽

Penghao Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

De Novo ◽

Fragment Size ◽

Gc Content ◽

Main Concern ◽

Whole Genome ◽

Assembly Quality ◽

Coverage Bias

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content.

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Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

10.17504/protocols.io.b3vgqn3w ◽

2022 ◽

Author(s):

Jason Nguyen ◽

Rebecca Hickman ◽

Tracy Lee ◽

Natalie Prystajecky ◽

John Tyson

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput ◽

Illumina Miseq ◽

Whole Genome ◽

Library Preparation ◽

Dna Libraries ◽

Reaction Volumes ◽

Single Size ◽

Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

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Contributions of de novo variants to systemic lupus erythematosus

European Journal of Human Genetics ◽

10.1038/s41431-020-0698-5 ◽

2020 ◽

Vol 29 (1) ◽

pp. 184-193 ◽

Cited By ~ 1

Author(s):

Jonas Carlsson Almlöf ◽

Sara Nystedt ◽

Aikaterini Mechtidou ◽

Dag Leonard ◽

Maija-Leena Eloranta ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Lupus Erythematosus ◽

De Novo ◽

Whole Genome ◽

Gene Promoters ◽

Single Nucleotide Variants ◽

Systemic Lupus ◽

Promoter Regions

AbstractBy performing whole-genome sequencing in a Swedish cohort of 71 parent-offspring trios, in which the child in each family is affected by systemic lupus erythematosus (SLE, OMIM 152700), we investigated the contribution of de novo variants to risk of SLE. We found de novo single nucleotide variants (SNVs) to be significantly enriched in gene promoters in SLE patients compared with healthy controls at a level corresponding to 26 de novo promoter SNVs more in each patient than expected. We identified 12 de novo SNVs in promoter regions of genes that have been previously implicated in SLE, or that have functions that could be of relevance to SLE. Furthermore, we detected three missense de novo SNVs, five de novo insertion-deletions, and three de novo structural variants with potential to affect the expression of genes that are relevant for SLE. Based on enrichment analysis, disease-affecting de novo SNVs are expected to occur in one-third of SLE patients. This study shows that de novo variants in promoters commonly contribute to the genetic risk of SLE. The fact that de novo SNVs in SLE were enriched to promoter regions highlights the importance of using whole-genome sequencing for identification of de novo variants.

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