Tag: PhiX

Special Episode 3: PhiX / UMIs / QC

Podcast: Explain Podcast Erschienen: 09.02.2024Dauer: 01:10:43 Getting the most out of Machines Chapters: 04:30 PhiX 14:30 low complexity 19:30 UMIs 32:10 FastQC 43:00 MultiQC 56:40 PycoQC PhiX concentrations for loading a validation run: knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001536 Dnatech on why UMIs are used: dnatech.genomecenter.ucdavis.edu/faqs/what-are-umis-and-why-are-they-used-in-high-throughput-sequencing/ BMH learning on UMIs: www.youtube.com/watch?v=sRPMsnhIBK0 FastQC for QC of…

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Endogenous Coriobacteriaceae enriched by a high-fat diet promotes colorectal tumorigenesis through the CPT1A-ERK axis

Bacteria Strain Cori.ST1911 was isolated from fresh stool of 20-week-old C57/BL6J mice fed a HFD at the Animal Center of the West China Hospital of Sichuan University. Briefly, faecal particles were ground with a glass grinding rod and suspended in sterile phosphate-buffered saline (PBS). After gradient dilution, the suspension was…

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Detection of DNA methylation signatures through the lens of genomic imprinting

Animals and samples The study included 10 pigs, 8 pigs were bred at the INRAE experimental farm (doi.org/doi.org/10.15454/1.5572415481185847E12) and 2 pigs come from breeding organizations in accordance with the French and European legislation on animal welfare. The animals belong to the same family, except for one LW animal. Animals were…

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Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses

INTRODUCTION Microbiome research, especially the detection of microorganisms by molecular techniques, has become a fundamental tool for investigating host-associated bacteria, such as those harbored by veterinary or human clinical samples[1,2]. Next-generation sequencing (NGS) approaches now enable the identification of slow-growing, non-cultivable, or non-viable bacteria contained in clinical specimens without relying…

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NGS Updates from ASHG: What’s New in Sequencing?

As expected, the NGS companies were sharing their news, out in full force, at the annual American Society for Human Genetics (ASHG) meeting last week in Washington, DC. Whether the updates came from the expo booths showcasing instruments, or users sharing data in the lecture halls, each company had progress…

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Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Construct design To transcribe and translate sequenced DNA clusters on an Illumina flow cell, our DNA constructs contained the following elements: a P5 adaptor, followed by a 28 nt unique barcode, a 27 nt unstructured spacer (5p UNS v2), a ribosome binding site, start codon, protein coding region, TolAk short linker, 2×…

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Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Soil incubation and cell extraction Approximately 3 kg of unmanaged marginal soil (pH 8) was collected from the Pacific Northwest National Laboratory field site in Prosser, Washington (46° 15′ 04″ N and 119° 43′ 43″ W) [23], homogenized, sieved (4 mm mesh size), and stored at 4 °C until further processing. Probe specific…

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DADA2 denoising not working (First return error 9 then 1 after increasing memory to 16 GB) – Technical Support

Did not find the exact answer to this on the forum; new to QIIME2, apologies. QIIME2-2023.5, running in Conda through university’s central Linux-based system Apply DADA2 denoising qiime dada2 denoise-paired –i-demultiplexed-seqs paired-end-demux.qza –p-trunc-len-f 280 –p-trunc-len-r 220 –p-n-threads 24 –o-representative-sequences rep-seqs-dada.qza –o-table table-dada2.qza –o-denoising-stats dada-stats.qza –verbose R version 4.2.3 (2023-03-15)Loading required…

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Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects

Bacterial strains, vectors and cloning E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacI q Z\(\Delta \)M15 Tn10 (Tet r)]), originally purchased from Stratagene (USA), was used for all phage display selections and Fab expression in screening. pEB32x6 phagemid vector (later “display vector”) was…

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DNA Sequencing | Element Biosciences

Avidity Sequencing™Scientists today have a wide variety of methods they can use to explore the how genomic changes or differences impact biology, including whole-genome or targeted sequencing, short or long reads, and numerous methods for assessing chromatin accessibility. By sequencing DNA in a fundamentally new way, the Element AVITI System…

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hypervariable region of 16s rrna

The mock community was created from pure cultures, whose 16S rRNA genes were determined through Sanger sequencing to be >3% different (Table S1). Bartram, Andrea. WebWhile 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species. Limited by sequencing Rarefaction…

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BBDuk Guide – DOE Joint Genome Institute

“Duk” stands to Decontamination Using Kmers. BBDuk was made to combine many common data-quality-related trimming, filtering, and masking actions into an single high-performance tool. It are capable of quality-trimming or filtering, adapter-trimming, contaminant-filtering via kmer matching, sequence masking, GC-filtering, length filtering, entropy-filtering, format conversion, histogram generation, subsampling, quality-score recalibration, kmer…

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DADA2 error from ITS sequences – Technical Support

Hello there, I’m working with ITS sequences from 45 samples in total. They were sequenced through an Illumina Novaseq 6000 instrument. Using the last version of QIIME2 (2023.5), my problem occurs while I perform the denoising analysis through dada2 with the following command: qiime dada2 denoise-paired –i-demultiplexed-seqs paired-end-demux.qza –p-trunc-len-f 155…

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Comparison of Oxford Nanopore Technologies and Illumina MiSeq sequencing with mock communities and agricultural soil

Study sites Soils were collected from two different sites (ARDEC: Colorado State University’s Agricultural Research, Development and Education Center in Fort Collins, CO; and CPCRC: USDA Columbia Plateau Conservation Research Center in Pendleton, OR). At each site, four replicate plots of no-till corn (ARDEC) or no-till annual wheat (CPCRC) were…

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Whole-genome sequencing of Listeria monocytogenes isolated from the first listeriosis foodborne outbreak in South Korea

Introduction Although globalization has provided opportunities for consumers to enjoy a wide range of products and expanded global food trade, the complexity of the international food supply has contributed to an increase in foodborne outbreaks (Quested et al., 2010; Hussain and Dawson, 2013). Worldwide efforts have ensured food safety by…

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Phylogenomic analysis supports Mycobacterium tuberculosis transmission between humans and elephants

1. Introduction Tuberculosis (TB) is a significant global burden and is widely reported to be a major public health and economic problem, costing the world $617 billion between 2000 and 2015 and projected to cost $1 trillion between 2015 and 2030 (1). It is the second leading cause of death…

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Assigned taxonomy after dada2 only “NAs” or “Mitochondria”

Hi, I followed the dada2 tutorial on 16S and ITS amplicon metagenomic data from soil samples. However, when trying to assign a taxonomic rank to my ASV table using DECIPHER, I only get “NAs”. When using the dada2 native’s Bayesian method, I get “NA” or “Mitochondria” (family). For my 16S…

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Enabling accurate and early detection of recently emerged SARS-CoV-2 variants of concern in wastewater

Wastewater sample collection, RNA extraction, and sequencing Houston Water collected and provided weekly 24-hour time-weighted composite influent (raw wastewater) samples from 39 wastewater treatment plants (WWTPs) in Houston covering a service area of approximately 580 miles2 and serving over 2.3 million people. In total, 2637 samples were analyzed. Untreated wastewater…

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Shallow shotgun sequencing reduces technical variation in microbiome analysis

Participant selection and sample collection Informed consent was obtained for five adult volunteers. The study protocols were reviewed and approved by the Advarra Institutional Review Board (Advarra, Inc., Columbia, MD). All analyses were performed according to the relevant guidelines and regulations. Fecal collection was completed by self-sampling, which has proven…

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NGS: Sequence QC – Texas A&M HPRC

Back to Bioinformatics Main Menu Evaluation FastQC GCATemplates available: grace terra module spider FastQC After running FastQC via the command line, you can ssh to an HPRC cluster enabling X11 forwarding by using the -X option and view the images using the eog tool. From your desktop: ssh -X username@grace.hprc.tamu.edu From your FastQC working…

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High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0-10) spacer-linked target specific primers without PhiX spike-in

Background: Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis….

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Failed to calculate read length

fastq_screen error: Failed to calculate read length 1 Running it like this: fastq_screen –conf $FASTQ_SCREEN_DATA/fastq_screen.conf –aligner bowtie2 –threads 6 03dpf.filtered.tagged.unmapped-1.fasta.gz Runs into this error. I cannot seem to find any information about this. Using fastq_screen v0.15.1 Reading configuration from ‘/FastQ_Screen_Genomes/fastq_screen.conf’ Adding database Human Adding database Mouse Adding database Rat Adding…

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Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

1. Introduction Soil harbors high abundance and diversity of prokaryotic microorganisms, with 1 g of soil containing up to 10 billion microbial cells (Torsvik and Øvreås, 2002) and 103–106 phylotypes (Bickel and Or, 2020), including many microorganisms that play critical biogeochemical roles (Crowther et al., 2019). With continuous expansion of prokaryotic…

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Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

This research complies with all relevant ethical regulations approved by the Human Investigation Review Board of Albert Szent-Györgyi Clinical Centre of the University of Szeged and the National Biodiversity Authority (NBA) of India. Permission for the faecal sample collection was obtained from the Human Investigation Review Board of Albert Szent-Györgyi…

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Systematic and benchmarking studies of pipelines for mammal WGBS data in the novel NGS platform | BMC Bioinformatics

Comparison of read level and improving the mapping efficiency according to trimming Since the generation of high-quality WGBS data ultimately impacts the quantification and interpretation of Cs methylation levels, it is indispensable to monitor the raw data quality and interrogate the appropriate pre-processing step to cleanse data [1]. To avoid…

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