Dataset Information

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen.

ABSTRACT: Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

SUBMITTER: Yoshida Y

PROVIDER: S-EPMC6126485 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen.

Yoshida Yuki Y Konno Sayuri S Nishino Ryousuke R Murai Yumi Y Tomita Masaru M Arakawa Kazuharu K

Journal of visualized experiments : JoVE 20180715 137

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius duja ...[more]

PMID: 30059025

Similar Datasets

Project description:Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27% (Fusobacterium nucleatum) to 73% (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n = 7) and a local outbreak of Klebsiella aerogenes (n = 5). The number of protocol steps and time required were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

Dataset Information

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen.

Publications

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets