Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An Economical and Flexible Dual Barcoding, Two-Step PCR Approach for Highly Multiplexed Amplicon Sequencing.

ABSTRACT: In microbiome research, phylogenetic and functional marker gene amplicon sequencing is the most commonly-used community profiling approach. Consequently, a plethora of protocols for the preparation and multiplexing of samples for amplicon sequencing have been developed. Here, we present two economical high-throughput gene amplification and sequencing workflows that are implemented as standard operating procedures at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. These workflows are based on a previously-published two-step PCR approach, but have been updated to either increase the accuracy of results, or alternatively to achieve orders of magnitude higher numbers of samples to be multiplexed in a single sequencing run. The high-accuracy workflow relies on unique dual sample barcoding. It allows the same level of sample multiplexing as the previously-published two-step PCR approach, but effectively eliminates residual read missasignments between samples (crosstalk) which are inherent to single barcoding approaches. The high-multiplexing workflow is based on combinatorial dual sample barcoding, which theoretically allows for multiplexing up to 299,756 amplicon libraries of the same target gene in a single massively-parallelized amplicon sequencing run. Both workflows presented here are highly economical, easy to implement, and can, without significant modifications or cost, be applied to any target gene of interest.

SUBMITTER: Pjevac P

PROVIDER: S-EPMC8173057 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

Project description:High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.

| S-EPMC4503924 | biostudies-literature

Multiplex PCR Targeted Amplicon Sequencing (MTA-Seq): Simple, Flexible, and Versatile SNP Genotyping by Highly Multiplexed PCR Amplicon Sequencing.

Project description:Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated "multiplex PCR targeted amplicon sequencing (MTA-seq)," and applied it to the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data with that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.

| S-EPMC5876661 | biostudies-literature

Improved ClickTags enable live-cell barcoding for highly multiplexed single cell sequencing.

Project description:Click chemistry-enabled DNA barcoding of cells provides a universal strategy for sample multiplexing in single-cell RNA-seq (scRNA-seq). However, current ClickTags are limited to fixed samples as they only label cells efficiently in methanol. Herein, we report the development of a new protocol for barcoding live cells with improved ClickTags. The optimized reactions barcoded live cells without perturbing their physiological states, which allowed sample multiplexing of live cells in scRNA-seq. The general applicability of this protocol is demonstrated in diversified types of samples, including murine and human primary samples. Up to 16 samples across these two species are successfully multiplexed and demultiplexed with high consistency. The wide applications of this method could help to increase throughput, reduce cost and remove the batch effect in scRNA-seq, which is especially valuable for studying clinical samples from a large cohort.

| S-EPMC9347365 | biostudies-literature

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Project description:Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

| S-EPMC4914102 | biostudies-literature

Highly multiplexed single-cell quantitative PCR

Project description:We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15 %, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNA in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifest as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.

2018-01-31 | GSE102734 | GEO

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes.

Project description:BackgroundMitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.ResultsWith the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.ConclusionsThis method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

| S-EPMC10155392 | biostudies-literature

Highly multiplexed single-cell quantitative PCR.

Project description:We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.

| S-EPMC5788347 | biostudies-literature

An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data.

Project description:High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields.Supplementary informationThe online version contains supplementary material available at 10.1007/s42995-023-00182-1.

| S-EPMC10689312 | biostudies-literature

A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology.

Project description:Studies using genome-wide single nucleotide polymorphisms (SNPs) have become commonplace in genetics and genomics, due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been intensively investigated. For example, the ultrahigh-multiplexed amplicon sequencing, Ion AmpliSeq, has been used as a high-throughput genotyping method mainly for diagnostic purposes. Here, we designed a custom panel targeting 3,187 genome-wide SNPs of fugu, Takifugu rubripes, and applied it for genotyping farmed fugu to test its feasibility in aquaculture studies. We sequenced two libraries consisting of different pools of individuals (n = 326 each) on the Illumina MiSeq sequencer. Consequently, over 99% target regions (3,178 SNPs) were amplified and 2,655 SNPs were available after filtering steps. Strong correlation was observed in the mean depth of coverage of each SNP between duplicate runs (r = 0.993). Genetic analysis using these genotype data successfully detected the known population structure and the sex determining locus of fugu. These results show the method is superior in repeatability and flexibility, and suits genetic studies including molecular breeding, such as marker assisted and genomic selection.

| S-EPMC6502806 | biostudies-literature

Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells.

Project description:We describe droplet-assisted RNA targeting by single-cell sequencing (DART-seq), a versatile technology that enables multiplexed amplicon sequencing and transcriptome profiling in single cells. We applied DART-seq to simultaneously characterize the non-A-tailed transcripts of a segmented dsRNA virus and the transcriptome of the infected cell. In addition, we used DART-seq to simultaneously determine the natively paired, variable region heavy and light chain amplicons and the transcriptome of B lymphocytes.

| S-EPMC6378878 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data