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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Once created, DNA sequencing libraries usually undergo a sequencing process known as ‘sequencing by synthesis’. First, the DNA library sample is transferred to a flowcell which is placed in the sequencing platform. The single-stranded DNA (ssDNA) fragments then adhere to the flowcell via hybridisation of the adapter regions (as described directly above). The attached DNA strands are then amplified to create ‘clusters’ of the exact same ssDNA molecules – these represent the template strand. Sequencing by synthesis is then performed by the creation of a copy strand of DNA, where fluorescently labelled nucleotides of different colours are added in a stepwise manner, each giving off a different coloured fluorescence signal depending on the nucleotide (e.g. the four nucleotides have four different colours). Depending on the length of the DNA libraries, this process is repeated until the entire DNA length has been sequenced. At the end of the sequencing process, the original DNA fragment will now be computationally identified in a series of nucleotide sequences. These computational sequences are commonly referred to as sequencing reads.
Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Matthew Lanktree, Amirreza Haghighi, Xueweng Song, York Pei
In our laboratory, 500 ng to 1 ug of genomic DNA is submitted to the core sequencing facility at the Centre for Applied Genomics (Sick Children's Hospital, Toronto, Canada) for genomic library preparation and whole-genome sequencing. DNA samples are quantified using Qubit High Sensitivity Assay and sample purity checked by NanoDrop OD260/280 ratio. One-hundred nanograms of DNA is used as input material for library preparation with the Illumina TruSeq Nano DNA Library Prep Kit following the manufacturer's protocol. In brief, DNA is fragmented to 350 bp using sonication on a Covaris LE220 instrument; fragmented DNA is end-repaired, A-tailed and TruSeq Illumina adapters with overhang-T are added; and libraries are validated for size and absence of primer dimers using a Bioanalyzer DNA High Sensitivity chip and quantified by qPCR using Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). Validated libraries are pooled in equimolar quantities and paired-end sequenced on an Illumina HiSeq X platform following Illumina's recommended protocol to generate paired-end reads of 150-bases in length.
Next-Generation Sequencing (NGS) for Companion Diagnostics (CDx) and Precision Medicine
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Il-Jin Kim, Mendez Pedro, David Jablons
Like the Ion Torrent system, in the SOLiD system, DNA is amplified in an emulsion PCR. The constructed DNA library is enriched and then attached to a glass flowchip. A universal primer is attached and ligated into the library adaptors and fluorescent signal is emitted after three-base cleavage from the octamer containing di-base probes.25 With multiple “ligation-detection-cleavage” steps, the whole sequencing process is completed.
The gut microbiome and metabolome in kidney transplant recipients with normal and moderately decreased kidney function
Published in Renal Failure, 2023
Yang Lan, Duo Wang, Jiayang He, Hongji Yang, Yifu Hou, Wenjia Di, Hailian Wang, Xiangwei Luo, Liang Wei
Construction of DNA Library. The DNA library was constructed according to the Novogene Co., Ltd (Beijing, China) manufacturer’s instructions. NEBNext Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) was used to generate sequencing libraries. Index codes were added to each sample to identify attributes for each sequence. The qualified DNA samples were randomly sheared to a size of 350 bp by sonication. Then these fragments were end-repaired, ligated with Illumina sequencing adapters, and PCR amplified. AMPure XP system (Beckman Coulter, CA, USA) was used to purify the PCR products, Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the size distribution of DNA library and real-time PCR was used to quantify DNA libraries [15].
Genomic characterization of four Escherichia coli strains isolated from oral lichen planus biopsies
Published in Journal of Oral Microbiology, 2021
Huitae Min, Keumjin Baek, Ahreum Lee, Yeong-Jae Seok, Youngnim Choi
Whole-genome sequencing and assembly were performed at ChunLab, Inc. (Seoul, Korea). The concentration and purity of the gDNA samples were determined using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, USA). To generate libraries, 400 ng of gDNA was fragmented into approximately 550 bp using a M220 Focused-ultrasonicator (Covaris, Brighton, UK), and the fragmented DNA was quantified using a DNA 7500 kit (Agilent, Palo Alto, USA) and a Bioanalyzer 2100 instrument (Agilent). Libraries were then constructed using a TruSeq DNA Library LT kit (Illumina, San Diego, USA) according to the manufacturer’s protocol. Whole-genome sequencing was performed on an Illumina MiSeq platform with 2 × 300 bp paired-end reads in conjunction with an Illumina MiSeq Reagent Kit v3 (600-cycle) (Illumina).
Opportunistic bacteria confer the ability to ferment prebiotic starch in the adult cystic fibrosis gut
Published in Gut Microbes, 2019
Yanan Wang, Lex E.X. Leong, Rebecca L. Keating, Tokuwa Kanno, Guy C.J. Abell, Fredrick M. Mobegi, Jocelyn M. Choo, Steve L. Wesselingh, A. James Mason, Lucy D. Burr, Geraint B. Rogers
Metagenomic libraries were fragmented and indexed using Nextera XT DNA Library Prep Kit (Illumina Inc.), and Nextera XT Index kit (Illumina Inc.), respectively as per manufacturer’s instructions. Amplicon libraries were then sequenced on the Illumina HiSeq 2500 platform at the SAHMRI David R Gunn Genomics Suite using Illumina HiSeq SBS 2 x 125bp v4 kit (Illumina Inc.). The sequencing resulted in an average of 18,586,781 ± 4,793,413 reads per sample. Bioinformatic processing of shotgun metagenomic sequence data was performed as described previously74, with minor modifications (Supplementary Methods). Relative gene abundances were estimated by dividing the number of the gene-length normalized read counts for each gene by the total of reads from that sample that uniquely mapped to any gene in the catalogue. For analysis of functional capacity, genes annotated against the KEGG database were collapsed row-wise, based on their KEGG orthology identifiers, and summed for each pathway.