Extracted genomic DNA was sheared to 100–400 bp (mean distribution 150 bp) using an LE220 ultrasonicator (Covaris). Libraries were prepared (NEBNext Ultra II DNA Library preparation kit, New England Biolabs) using initial adaptor ligation and barcoding with unique dual indexed barcodes (Integrated DNA Technologies). Dual indexed samples were amplified (6 cycles of PCR, KAPA HiFi kit, Roche), quantified (Accuclear dsDNA Quantitation Solution, Biotium), then pooled in pre-assigned groups of 48 or 32 to generate equimolar pools on the basis of total DNA concentration. Pooled DNA (500 ng) was hybridized using 120-mer RNA baits (SureSelect Target enrichment system, Agilent technologies; bait design ELID ID 0616571)4 (link),37 (link). Enriched libraries were sequenced on Illumina HiSeq 4000 to generate 150 bp paired-end reads at the Wellcome Sanger Institute (UK) as previously described38 . For one rabbit-passaged sample from Melbourne, Australia (TPA_AUSMELT-1)30 , genomic DNA extracted from historically archived tissue lysate was sequenced on Illumina NextSeq 500 (150 bp paired-end reads, Nextera DNA Flex libraries) without any previous enrichment to an estimated 1 Gb per sample at the Doherty Institute (Australia).
Beale M.A., Marks M., Cole M.J., Lee M.K., Pitt R., Ruis C., Balla E., Crucitti T., Ewens M., Fernández-Naval C., Grankvist A., Guiver M., Kenyon C.R., Khairullin R., Kularatne R., Arando M., Molini B.J., Obukhov A., Page E.E., Petrovay F., Rietmeijer C., Rowley D., Shokoples S., Smit E., Sweeney E.L., Taiaroa G., Vera J.H., Wennerås C., Whiley D.M., Williamson D.A., Hughes G., Naidu P., Unemo M., Krajden M., Lukehart S.A., Morshed M.G., Fifer H, & Thomson N.R. (2021). Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis. Nature Microbiology, 6(12), 1549-1560.
Bp 400 Dna libraries Dsdna GenomicLigation Rabbit Tissue
Corresponding Organization : London School of Hygiene & Tropical Medicine
Other organizations :
University College London Hospitals NHS Foundation Trust, University College London, Hospital for Tropical Diseases, BC Centre for Disease Control, MRC Laboratory of Molecular Biology, University of Cambridge, National Public Health and Medical Officer Service, Instituut voor Tropische Geneeskunde, Leeds General Infirmary, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Sahlgrenska University Hospital, Manchester Royal Infirmary, Kazan Federal University, Vall d'Hebron Hospital Universitari, University of Washington, Seattle University, Tuvan State University, Ministry of Health of the Russian Federation, Leeds Teaching Hospitals NHS Trust, Colorado School of Public Health, University of Colorado Denver, Midland Regional Hospital Portlaoise, Alberta Hospital Edmonton, Queen Elizabeth Hospital Birmingham, Institute of Environmental Science and Research, University of Queensland, Peter Doherty Institute, University of Sussex, Brighton and Sussex Medical School, University of Gothenburg, University of Alberta, Örebro University, University of British Columbia
Shearing of genomic DNA to 100–400 bp (mean distribution 150 bp) using an LE220 ultrasonicator (Covaris)
Library preparation using NEBNext Ultra II DNA Library preparation kit and unique dual indexed barcodes (Integrated DNA Technologies)
Amplification of dual indexed samples with 6 cycles of PCR using KAPA HiFi kit (Roche)
Pooling of amplified samples in pre-assigned groups of 48 or 32 to generate equimolar pools based on total DNA concentration
Hybridization of pooled DNA (500 ng) using 120-mer RNA baits (SureSelect Target enrichment system, Agilent technologies; bait design ELID ID 0616571)
dependent variables
Sequencing of enriched libraries on Illumina HiSeq 4000 to generate 150 bp paired-end reads
Sequencing of one rabbit-passaged sample (TPA_AUSMELT-1) on Illumina NextSeq 500 (150 bp paired-end reads, Nextera DNA Flex libraries) without any previous enrichment to an estimated 1 Gb per sample
control variables
Input DNA concentration (500 ng) for hybridization using 120-mer RNA baits
Library preparation kit (NEBNext Ultra II DNA Library preparation kit)
Amplification kit (KAPA HiFi kit)
Sequencing platform (Illumina HiSeq 4000 and Illumina NextSeq 500)
Annotations
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