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50 results for “Library preparation”
Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles
<p>We aimed to simplify our fetal <i>RHD</i> genotyping protocol by changing the method to attach Illumina's sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its quantitative accuracy by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal <i>RHD</i> genotyping for East Asian populations into clinical practice.</p>
Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles
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Cost-effective library preparation for whole genome sequencing with feather DNA
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Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples
Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of biodiversity, diet and ecological interactions as its inherent labelling of amplicons allows sequencing of taxonomically informative genetic markers from many samples in parallel. However, the occurrence of so-called 'tag-jumps' can cause incorrect assignment of sequences to samples and artificially inflate diversity. Two steps during library preparation of pools of 5' nucleotide-tagged amplicons have been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step and ii) post-ligation PCR amplification of amplicon libraries. The discovery of tag-jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin-tags to ensure that tag-jumps cannot result in false assignments of sequences to samples. As this increases both cost and workload, a metabarcoding library preparation protocol which circumvents the two steps that causes tag-jumps is needed. Here, we demonstrate Tagsteady, a metabarcoding Illumina library preparation protocol for pools of nucleotide-tagged amplicons that enables efficient and cost-effective generation of metabarcoding data with virtually no tag-jumps. We use pools of twin-tagged amplicons to investigate the effect of T4 DNA polymerase blunt-ending and post-ligation PCR on the occurrence of tag-jumps. We demonstrate that both blunt-ending and post-ligation PCR, alone or together, can result in detrimental amounts of tag-jumps (here, up to ca. 49% of total sequences), while leaving both steps out (the Tagsteady protocol) results in amounts of sequences carrying new combinations of used tags (tag-jumps) comparable to background contamination.
Supplementary Figure Legend S1: An overview of DNA samples included in the NGS based chimerism validation process and AlloSeq HCT library preparation workflow.
<p><strong>Supplementary Figure </strong><strong>Legend S1: </strong>An overview of DNA samples included in the NGS based chimerism validation process and AlloSeq HCT library preparation workflow. <strong>S1a: </strong>An overview of clinical and artificial DNA included in the NGS based chimerism validation process (Clinical samples, Artificial construct samples and ASHI EMO Proficiency samples). <strong>S1b:</strong> Schematic presentation of time required for the NGS Chimerism assay. <strong>S1c.</strong> AlloSeq HCT library preparation workflow.</p>
Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples
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Data from: BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction
Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing inherent properties of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.
Data from: The impact of library preparation protocols on the accuracy of allele frequency estimates in Pool-Seq data
Sequencing pools of individuals (Pool-Seq) is a cost-effective method to determine genome-wide allele frequency estimates. Given the importance of meta-analyses combining data sets, we determined the influence of different genomic library preparation protocols on the consistency of allele frequency estimates. We found that typically no more than 1% of the variation in allele frequency estimates could be attributed to differences in library preparation. Also read length had only a minor effect on the consistency of allele frequency estimates. By far, the most pronounced influence could be attributed to sequence coverage. Increasing the coverage from 30- to 50-fold improved the consistency of allele frequency estimates by at least 27%. We conclude that Pool-Seq data can be easily combined across different library preparation methods, but sufficient sequence coverage is key to reliable results.
Data from: BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction
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Data from: The impact of library preparation protocols on the accuracy of allele frequency estimates in Pool-Seq data
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Data from: A fast and efficient single-stranded genomic library preparation method optimized for ancient DNA
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Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [WGBS]
GEO Series GSE128731. Homo sapiens. 41 samples. Type: Methylation profiling by high throughput sequencing.
Optimized ATAC-seq library preparation from Drosophila neurons
GEO Series GSE197760. Drosophila melanogaster. 5 samples. Type: Genome binding/occupancy profiling by high throughput sequencing.
Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol
GEO Series GSE101520. Homo sapiens. 87 samples. Type: Expression profiling by high throughput sequencing.
Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input
GEO Series GSE124167. Mus musculus. 18 samples. Type: Expression profiling by high throughput sequencing.
Evaluation of RNA amplification and RNA-Seq library preparation protocols for spermatozoa RNA profiling
GEO Series GSE57503. Homo sapiens. 16 samples. Type: Expression profiling by high throughput sequencing.
Dts-seq: a simple method of library preparation for a highly reproducible characterization of the tRNA epitranscriptome by deep sequencing
GEO Series GSE135937. Escherichia coli str. K-12 substr. MG1655. 15 samples. Type: Non-coding RNA profiling by high throughput sequencing.
Systematic assessment of commercially available low-input miRNA library preparation kits
GEO Series GSE133719. Homo sapiens; synthetic RNA. 126 samples. Type: Non-coding RNA profiling by high throughput sequencing.
Sequencing of small RNA libraries prepared from Invertebrate Iridescent Virus 6 (IIV6)-infected S2 cells or adult flies.
GEO Series GSE41007. Invertebrate iridescent virus 6. 4 samples. Type: Non-coding RNA profiling by high throughput sequencing.
Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing
GEO Series GSE128734. Homo sapiens. 53 samples. Type: Methylation profiling by high throughput sequencing; Methylation profiling by array.
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International Brain Laboratory public data
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OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.