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Introduction to Genomics
Published in Altuna Akalin, Computational Genomics with R, 2020
The sequencing technology is still evolving. Obtaining longer single-molecule reads, and preferably, being able to call base modifications on the fly is the next frontier. With longer reads, the genome assembly will be easier for the regions that have high repeat content. With single-molecule sequencing, we will be able to tell how many transcripts are present in a given cell population without relying on fragment amplification methods which can introduce biases.
DNA Methods in Veterinary Medicine
Published in Rebecca A. Krimins, Learning from Disease in Pets, 2020
Lastly, another approach to genome assembly is the use of Hi-C, so named because it uses high-throughput sequencing on chromatin. Hi-C is a form of chromatin confirmation techniques. It uses the way that DNA is wound around chromatin to gain information about that DNA. In this method, chromatin is isolated with the bound DNA. Formaldehyde is added, which causes the different pieces of DNA that are close to each other to link together. More links will occur with nearby sequences than distant ones. After the formaldehyde step, the DNA is digested with one or more restriction enzymes, which leaves the linked DNA pieces stuck together. Those linked fragments are then ligated together to form chimeric molecules whose ends originate at different positions along the chromosome (Dudchenko et al., 2018). After standard paired-end Illumina sequencing the number of near to distant joins can be determined by comparing the reads to scaffolds generated by other methods described above. Hi-C has been used to assemble genomes for dozens of animals (e.g., Humble et al., 2019; see also, DNAzoo.org).
Gene Expression Profiling to Detect New Treatment Targets in Leukemia and Lymphoma: A Future Perspective
Published in Gertjan J. L. Kaspers, Bertrand Coiffier, Michael C. Heinrich, Elihu Estey, Innovative Leukemia and Lymphoma Therapy, 2019
Torsten Haferlach, Wolfgang Kern, Alexander Kohlmann
In addition to all the sequences represented on the HG-U133A and HG-U133B two-array set, the HG-U133 Plus 2.0 microarray also covers 9921 new probe sets representing approximately 6500 new genes. These gene sequences were selected from GenBank, dbEST, and RefSeq. Sequence clusters were created from the UniGene database (Build 159, January 25, 2003) and refined by analysis and comparison with a number of other publicly available databases, including the Washington University EST trace repository and the NCBI human genome assembly Build 31 (www.affymetrix.com). Thus, in using this comprehensive whole human genome expression array, an extensive coverage of the human genome is reached. HG-U133 Plus 2.0 microarrays are manufactured as standard format arrays with more than 54,000 probe sets of a feature size of 11 µm and use 11 probe pairs per sequence. The oligonucleotide length is 25 mer.
The evolution and competitive strategies of Akkermansia muciniphila in gut
Published in Gut Microbes, 2022
Ji-Sun Kim, Se Won Kang, Ju Huck Lee, Seung-Hwan Park, Jung-Sook Lee
Genomic DNA was extracted from cells grown on TSAB as described previously.58 Whole-genome sequencing of the A. muciniphila strains was performed using PacBio RS II single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences). A standard PacBio library with an average of 20 kb inserts were prepared and were sequenced. De novo assembly was conducted using the hierarchical genome-assembly process (HGAP) pipeline of the SMRT Analysis v2.3.0. In order to correct sequencing errors that can occur at both ends of a contig, the SMRT resequencing protocol was performed with assembly that the first half of the contig was switched with the second half. As the result of assembly, A. muciniphila KGMB strains and type strain KCTC 15667 T had complete circular genome sequences as described in Table 1.
Interleukin-1β secretion induced by mucosa-associated gut commensal bacteria promotes intestinal barrier repair
Published in Gut Microbes, 2022
Wan-Jung H. Wu, Myunghoo Kim, Lin-Chun Chang, Adrien Assie, Fatima B. Saldana-Morales, Daniel F. Zegarra-Ruiz, Kendra Norwood, Buck S. Samuel, Gretchen E. Diehl
Fecal pellets from AVMN-treated mice were resuspended in PBS to 100 mg/ml and dilutions were plated on blood agar plates (Fisher) and cultured overnight at 37°C under normal or anaerobic conditions using BD GasPak (Fisher). For sequencing, genomic DNA was extracted with phenol chloroform, and DNA was sheared to 15kb using Covaris g-TUBE® devices, allowing for sizes 5 kb and larger. The library preparation was carried out using SMRTbell Template kit 1.0 Exo VII protocol and the sample was barcoded with PacBio Adaptor. Genome sequencing was performed using the Pacific Biosciences Sequel sequencing platform. Long reads were assembled de novo into two contigs (main chromosome and 1 plasmids) using Canu (v. 1.6).73 Gene prediction and annotation were carried out using the webservice PATRIC.74 Genomic visualization was performed using Circos v0.69–9.75 Genomic comparison was done using the PATRIC webservice and phylogenomic reconstructions were done using the GToTree pipeline and its associated dependencies.76–80 Sequencing reads and the genome assembly were submitted to NCBI under the bioproject PRJNA725420.
Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis
Published in Gut Microbes, 2022
Maximilian Baumgartner, Rebecca Zirnbauer, Sabine Schlager, Daniel Mertens, Nikolaus Gasche, Barbara Sladek, Craig Herbold, Olga Bochkareva, Vera Emelianenko, Harald Vogelsang, Michaela Lang, Anton Klotz, Birgit Moik, Athanasios Makristathis, David Berry, Stefanie Dabsch, Vineeta Khare, Christoph Gasche
Bacterial DNA was extracted using a phenol chloroform-based method. Whole-genome sequencing was performed using HiSeqV4 PE125 methodology. For genome assembly, the spades pipeline was used. Assemblies were submitted to NCBI for annotation. The CFSAN SNP pipeline was used with the E. coli reference genome O103:H2 12009 to construct an SNP matrix with the 57 strains from this study and 348 publicly available AEEC genomes of diverse pathotypes and one E. albertii genome. For phylogenomic maximum likelihood inference, IQ-TREE was applied with the best-fit model automatically selected by ModelFinder.41 For pangenome analysis, the Roary pipeline was used with standard parameters, followed by Scoary for the identification of associations between all genes in the accessory genome and EspG2 and EspV positivity.42 Pangenome composition was visualized with Phandango. To investigate the presence of known virulence factors, the VFDB database was used. For the detection of novel hypothetical secreted proteins and additional secretion systems, the EffectiveDB was applied. Pairwise comparison between EspG2-pos and EspV-pos genomes was performed with the Mann–Whitney U test, prevalences were compared using Fisher’s exact test, with Bonferroni correction for multiple comparisons. Sequencing data and assemblies are publicly accessible at NCBI under the project number PRJNA528578. Additional information on bioinformatic analysis can be found in the supplementary method section.