Faculty Opinions recommendation of Scalable whole-genome single-cell library preparation without preamplification.

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.

Download Full-text

SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

Nucleic Acids Research ◽

10.1093/nar/gkw1110 ◽

2016 ◽

Vol 45 (6) ◽

pp. e36-e36 ◽

Cited By ~ 17

Author(s):

Amanda Raine ◽

Erika Manlig ◽

Per Wahlberg ◽

Ann-Christine Syvänen ◽

Jessica Nordlund

Keyword(s):

Preparation Method ◽

Whole Genome ◽

Library Preparation ◽

Bisulphite Sequencing ◽

Library Preparation Method

Download Full-text

Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers

Lab on a Chip ◽

10.1039/c7lc00430c ◽

2017 ◽

Vol 17 (15) ◽

pp. 2619-2630 ◽

Cited By ~ 1

Author(s):

Gordon D. Hoople ◽

Andrew Richards ◽

Yan Wu ◽

Kota Kaneko ◽

Xiaolin Luo ◽

...

Keyword(s):

Transcriptome Sequencing ◽

Whole Genome ◽

Library Preparation ◽

Low Input ◽

Dna And Rna

Gel-seq enables researchers to simultaneously prepare libraries for both DNA and RNA from 100 cells.

Download Full-text

MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.563 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii408-iii408

Author(s):

Marina Danilenko ◽

Masood Zaka ◽

Claire Keeling ◽

Stephen Crosier ◽

Rafiqul Hussain ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Copy Number ◽

Single Cell Analysis ◽

Mutational Analysis ◽

Single Cells ◽

Clonal Evolution ◽

Whole Genome ◽

Clinically Significant

Abstract Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.

Download Full-text

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

PLoS ONE ◽

10.1371/journal.pone.0254971 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0254971

Author(s):

Federico Rossi ◽

Alessandro Crnjar ◽

Federico Comitani ◽

Rodrigo Feliciano ◽

Leonie Jahn ◽

...

Keyword(s):

Dna Methylation ◽

Tree Ring ◽

High Throughput ◽

Environmental Conditions ◽

High Throughput Sequencing ◽

Wood Formation ◽

Whole Genome ◽

Library Preparation ◽

Bisulfite Treatment ◽

Standing Trees

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

Download Full-text