Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

SLUPT: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template Protocol

SLUPT stands for Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, U-containing Template. SLUPT enables the user to create a DNA library, and/or multiple mutations of a given DNA target, independently or simultaneously. In particular, when SLUPT is used in conjunction with known structural and sequence homology data, one can create highly directed libraries/mutations spread across the gene of interest.

Shared and Distinct Genetic Features in Human and Canine B-Cell Lymphomas

Introduction Animal models of human cancers are an important tool for the development and preclinical evaluation of new treatments. Canine B-cell lymphoma (cBCL) is an appealing alternative to murine preclinical models due to its frequent, spontaneous incidence and its clinical and histological similarity to human B-cell non-Hodgkin lymphoma (NHL). The potential utility of cBCL as a veterinary model of human B-cell lymphomas would be bolstered by a more complete understanding of the genetic features found in cBCL. Methods To study the genetics of cBCL, we obtained fresh frozen and matched plasma/serum from 86 patients from the Canine Comparative Oncology Genomic Consortium(CCOGC) with 65 confirmed as B-cell lymphomas by immunophenotyping. Tumor DNA was prepared into libraries using the QIAseq FX DNA Library Kit (Qiagen). Plasma and serum DNA was prepared into libraries using the NebNext Ultra II DNA Library Prep Kit. Targeted hybridization enrichment was performed on the libraries using our custom baits and sequencing reads were aligned to canFam3.1 using Geneious and each mutation was visually confirmed. Variants were annotated with Variant Effect Predictor and human-dog pairwise alignments were extracted from Ensembl to identify the orthologous human amino acid for all canine variants. Results Our analysis confirmed the previously reported high frequency of mutations in TRAF3 and FBXW7. We also observed mutations in POT1, TP53, and SETD2 at similar frequencies to those reported in previous studies. DDX3X was mutated in 20% of cases, which is substantially higher than previously reported. MYC mutations were also more frequent (13%) than has been previously described in cBCL. In human lymphomas, MYC is commonly deregulated by translocation to a potent enhancer and these events are often associated with point mutations in MYC that are induced by aberrant somatic hypermutation (aSHM). Interestingly, we identified a more focal pattern of MYC mutations in cBCL that implies they do not result from aSHM and are likely functional. This finding implicates the conserved MYC phosphodegron sequence, a motif commonly mutated among additional aSHM-associated mutations, as the target of bona fide driver mutations in both human and cBCLs. Mutations in FBXW7 primarily affected the substrate recognition domain responsible for MYC degradation. The observation that MYC and FBXW7 mutations did not co-occur in any canine patient is consistent with the notion that FBXW7 mutations operate as an alternative path to MYC stabilization which is not frequently observed in human NHL. DDX3X was one of the most frequently mutated genes in our cohort (20%). DDX3X mutations are common in human Burkitt lymphoma and, though less abundant in hDLBCL, tend to be observed in samples with MYC translocations. In Burkitt lymphoma, these mutations display a sex-specific pattern, wherein females show mainly missense mutations, while males are affected by loss-of-function mutations. Interestingly, all DDX3X mutations in cBCL are missense variants and are presumed to be dominant acting. This lack of sex difference in DDX3X mutations is an important distinction between human and canine B-cell lymphomas that warrants further exploration. Conclusions Our study has revealed key differences in the mutational profiles of canine and human B-cell lymphomas and provides an impetus for enhanced genomic characterization of canine lymphomas as a model for human NHL, particularly in clinical trial settings. Disclosures Grande: Sage Bionetworks: Current Employment. Alcaide: GA Diagnostics AB: Current Employment. Morin: Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Epizyme: Patents & Royalties. Coyle: Allakos, Inc.: Consultancy.

Diagnosis of Hordeum vulgare Genomic Profile: Review

This review represent plants genetic diversity (PDG) generally in crop plant and especially in Barley (Hordeum vulgare), can be studied (PDG) and stored as a (PGR) plant genetic resources as gene bank , DNA library for saved genetic material at long time and crops improvement can be utilized in breeding programs strategies in future. In this study observed the significance of plant genetic diversity (PGD) and (PGR) especially on agriculturally important crops , analysis of plant genomic using molecular markers. Barley is a well important studies crops using as a model for study genetic plant, cultivated barley Hordeum vulgare easily hybridization by genetic fingerprinting with wiled barley Hordeum spontaneum. The molecular markers showed their relation with locus of geographic factors and imposed stresses. Here, discussed barley genomic through relationship between genotype and phenotype traits using molecular markers useful for genetic physiological maps construction.

Optimization of Enzymatic Fragmentation is Crucial To Maximize Genome Coverage: A Comparison of Library Preparation Methods for Illumina Sequencing

Background Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 µg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits. Results We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits` fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection. Conclusions The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Mitochondrial DNA sequencing of a wet-collection syntype demonstrates the importance of type material as genetic resource for lantern shark taxonomy (Chondrichthyes: Etmopteridae)

After initial detection of target archival DNA of a 116-year-old syntype specimen of the smooth lantern shark, Etmopterus pusillus , in a single-stranded DNA library, we shotgun-sequenced additional 9 million reads from this same DNA library. Sequencing reads were used for extracting mitochondrial sequence information for analyses of mitochondrial DNA characteristics and reconstruction of the mitochondrial genome. The archival DNA is highly fragmented. A total of 4599 mitochondrial reads were available for the genome reconstruction using an iterative mapping approach. The resulting genome sequence has 12 times coverage and a length of 16 741 bp. All 37 vertebrate mitochondrial loci plus the control region were identified and annotated. The mitochondrial NADH2 gene was subsequently used to place the syntype haplotype in a network comprising multiple E. pusillus samples from various distant localities as well as sequences from a morphological similar species, the shortfin smooth lantern shark Etmopterus joungi . Results confirm the almost global distribution of E. pusillus and suggest E. joungi to be a junior synonym of E. pusillus . As mitochondrial DNA often represents the only available reference information in non-model organisms, this study illustrates the importance of mitochondrial DNA from an aged, wet collection type specimen for taxonomy.

SCREENING OF C-DNA LIBRARY USING COLONY PCR

The principal of cdna strand was that it was combined from complete rna utilizing an oligo(dt)- containing preliminary. After oligo(dg) following the absolute cdna was enhanced by pcr utilizing two groundworks correlative to oligo(da) and oligo(dg) closures of the cdna beginning from 10 j558lμm3 myeloma cells, absolute cdna was incorporated and intensified roughly 10 5 overlay. A library containing 10 6 clones was set up from 1/6 of the enhanced cdna. Screening of the library with tests for three qualities communicated in these cells uncovered various comparing clones for each situation. The longest acquired clones contained supplements of 1. 5kb length. No arrangements starting from transporters or from rrna was found in 14 haphazardly picked clones.

Recovery of small plasmid sequences via Oxford Nanopore sequencing

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

FindingNemo in OneDay: Ultra-Long ONT Library Preparation from Cell to Flowcell in One Day v1

This protocol is focussed on the isolation of ultra-high molecular weight (UHMW) DNA, library preparation and clean up ready for sequencing on the Oxford Nanopore Technology (ONT) platforms, all within one working day. This protocol is optimised for human cell lines. Summary of the workflow:

SARS-CoV-2 DNA library preparation using an adapted version of Illumina DNA prep protocol v.1.1 v1

This protocol describes the steps to prepare DNA libraries from PCR amplicons using the Illumina DNA prep library kit. The current library preparation protocol was adapted from the original Illumina DNA Prep Reference Guide (document #1000000025416 v09) using low input DNA samples as the starting material. We omitted laboratory equipment such as the Eppendorf 96-well PCR plate, microseal adhesive seals. In addition, we replaced 96-well plate magnetic stand with a magnetic stand suitable for 1.5 ml tubes. The added value of this protocol is that PCR reactions happen in 0.2 ml PCR tubes, and it can be implemented without a separate purchase of 96-well PCR plates or a magnetic stand for PCR plates. Using individual 0.2 ml tubes increases the user flexibility when running few samples for library preparation. The other advantage of using our adapted protocol is the reduced library preparation cost when running few specimens. For instance, implementing the current protocol might be cheaper than the original Illumina protocol when using the Illumina® DNA Prep, (M) Tagmentation (24 Samples) catalog number 20018704. The protocol has proven effective for processing hundreds of DNA libraries from tiled virus amplicons such as Sapovirus and SARS-CoV-2 submitted to public repositories such as GISAID and GenBank.