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opencc-zeroJun 2020View details →

Data from: More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Non-invasive genotyping methods have become key elements of wildlife research over the last two decades, but their widespread adoption is limited by high costs, low success rates, and high error rates. The information lost when genotyping success is low may lead to decreased precision in animal population densities, which could misguide conservation and management actions. Single nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to genotyping errors, and produce results that are easily shared among laboratories. Here, we outline a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis latrans) using 26 SNPs. <a name="_Hlk33181599">Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, representing a substantial increase compared to previous microsatellite-based studies while remaining at a low cost of under $5 per PCR replicate (excluding labor). </a>The accuracy of the genotypes was further corroborated in that genotypes from scats matching known, GPS-collared coyotes were always located within the territory of the known individual. We also show that different levels of multiplexing produced similar results, but that PCR product cleanup strategies can have substantial effects on genotyping success. By making noninvasive genotyping more affordable, accurate, and efficient, this research may allow for a substantial increase in the use of noninvasive methods to monitor and conserve free-ranging wildlife populations.

opencc-by-4.0Jul 2020View details →

Amplicon sequencing of endolithic microbial communities of Joshua Tree National Park, California (USA)

ITS and 16S amplicon sequencing data of ten rock samples, colonized by endolithic communities, collected at Joshua Tree National Park (May 2016). The Ribosomal Internal Transcribed Sequence 1 region (ITS1) and V4 region of the 16S rRNA gene were amplified using barcoded ITS1F/ITS2 and F515/R806 primers, respectively, according to Earth Microbiome Project's protocols. Fungal ITS and bacterial 16S primers were made available through the Alfred P. Sloan Foundation Built. Sequencing (2x300 bp) has been performed on Illumina Miseq platform at University of California Riverside (UCR). Data analyses were performed on the High-Performance Computing Cluster at UCR in the Institute of Integrative Genome Biology. The dataset consists of 2 zip archives. Here is a summary of their contents: <ul> <li>ITS_fungi: R1.fastq and R2.fastq files</li> <li>16S_bacteria: R1.fastq and R2.fastq files</li> </ul>

opencc-zeroAug 2020View details →

An amplicon sequencing protocol for attacker identification from DNA traces left on artificial prey

<ol> <li>Clay model studies are a popular tool to identify predator-prey interactions that are challenging to observe directly in the field. But despite its wide use, the method's applicability is limited by its low taxonomic resolution. Attack marks on clay models are usually identified visually, which only allows classification into higher taxonomic levels of predators. Thus, the method is often biased, lacks proof and, above all, standardization.</li> <li>Here, we tested whether precise identification of attackers can be provided by amplification and sequencing of mitochondrial DNA left in bite marks on clay models. We validated our approach in a controlled laboratory study as well as in a field experiment using clay models of a common European amphibian, the European fire salamander (Salamandra salamandra). DNA based taxonomic assignments were additionally compared to visual assessments of bite marks.</li> <li>We show that trace DNA of attackers can be routinely isolated and sequenced from bite marks, providing accurate species level classification. In contrast, visual identification alone yielded a high number of unassigned predator taxa. We also highlight the sensitivity of the method and show likely sources of contamination as well as probable cases of secondary and indirect predation.</li> <li>Our standardized approach for species level attacker identification opens up new possibilities far beyond the standard use of clay models to date, including food web studies at unprecedented detail, invasive species monitoring as well as biodiversity inventories.</li> </ol>

opencc-by-4.0Aug 2020View details →

PacBio amplicon re-sequencing of 62 P. tricornutum genomic loci – processed datasets

Processed datasets from PacBio amplicon re-sequencing of 62 P. tricornutum genomic loci. Raw data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA658511.  Available datasets:  - reference file for regions selected for amplicon sequencing: Phaeodactylum_tricornutum_amplicon_sequencing_loci.fa - final .bam files containing processed PacBio sequencing reads aligned to the reference: PacBio_amplicon_seq_T1.bam  and PacBio_amplicon_seq_T6.bam  - . table files with the position, reference and alternative allele for reliable biallelic SNPs selected in ILLUMINA sequencing of the culture at T1:  P_tricornutum_PacBio_amplicon_sequencing_T1_SNPs.table and P_tricornutum_PacBio_amplicon_sequencing_T6_SNPs.table Re-sequencing of 62 endogenous P. tricornutum loci selected in a genome-wide analysis of haplotype diversity. The goal was to determine the number of haplotypes per locus and the appearance of new haplotypes over time. The length of the sequenced loci was 2kb (+/- 5%).  Loci were amplified by emulsion PCR on the same culture harvested in two time points: five loci were amplified one month (T1) and all loci were amplified 6 months (T6) after the start of the culture from a single cell. Plasmids containing cloned GFP or YFP were amplified separately as a control for random errors. Control reactions for artificial haplotypes detection consisted of mixed CFP with YFP or CFP with GFP. Amplicons were pooled together into two samples. Sample PacBio_AS_T1 contained five P. tricornutum endogenous amplicons from DNA harvested at T1 time point, GFP amplified separately and CFP+YFP amplified in one reaction. Sample PacBio_AS_T6 contained 63 P. tricornutum endogenous amplicons from DNA harvested at T6 time point, YFP amplified separately and CFP+GFP amplified in one reaction. Samples were mixed in 1:9 PacBio_AS_T1: PacBio_AS_T6 ratio before sequencing on one PacBio Sequel SMRT cell.

opencc-zeroSep 2020View details →

Data from: Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 x 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

opencc-zeroDec 2012View details →

Data from: Quantifying sequence proportions in a DNA-based diet study using Ion Torrent amplicon sequencing: which counts count?

A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high-throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals' diet. Seals were fed fish in fixed proportions, a chordate-specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM™. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high-throughput sequencing will require careful experimental design and thoughtful data analysis.

opencc-zeroDec 2014View details →

Data from: From benchtop to desktop: important considerations when designing amplicon sequencing workflows

Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose.

opencc-zeroDec 2012View details →

Data from: Amplicon pyrosequencing late Pleistocene permafrost: the removal of putative contaminant sequences and small-scale reproducibility

DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small-scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi-squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present-day Yukon.

opencc-by-4.0Jun 2017View details →

Pre-processed B-cell receptor amplicon sequencing data from SRR1842411

An example dataset containing B-cell receptor (BCR) gene sequences. This dataset is intended to be used for testing software tools developed to annotate (i.e. map Variable, Diversity and Joining segments) and perform clonal analysis of BCR sequencing data. Sequencing: Libraries prepared using 5'RACE from PBMCs of a healthy donor. Input molecules were tagged with unique molecular identifiers (UMIs). Sequencing was ran on MiSeq , 300+300bp reads. Contents: The dataset contains both raw sequencing reads and high-quality consensus sequences assembled using unique molecular tagging (UMI) approach. Consensus assembly corrects for sequencing errors and eliminates sequencing artifacts. <ul> <li>age_ig_s7_R1.fastq.gz and age_ig_s7_R2.fastq.gz contain raw reads</li> <li>age_ig_s7_R1.t10.cf.fastq.gz and age_ig_s7_R2.t10.cf.fastq.gz contain consensus sequences</li> </ul> All files contain an UMI tag sequence in their header, in form UMI:NNNN:QQQQ where N is the base character and Q is the quality character (for assembled consensuses the total number of reads is given instead of Q string). Note that consensus sequences were assembled using only raw sequences that correspond to UMI tags supported by at least 10 sequencing reads. That means that consensus sequence files contain a subset of all UMI tags found in raw sequences. Thus, if one wants to assess software performance on raw sequencing reads using assembled consensus sequences as a high-quality data standard, raw sequencing reads should be filtered to contain only those UMI tags that are present in consensus sequence file. Citations: The whole dataset was used to benchmark MiXCR software and was originally referenced in Bolotin DA, et al. MiXCR: software for comprehensive adaptive immunity profiling Nature methods 12(5):380-381, 2015. Data pre-processing was carried out using MIGEC software, Shugay M et al. Towards error-free profiling of immune repertoires. Nature Methods 11(6):653-655, 2014. Contributors: The dataset was generated in Prof. Chudakov lab (Adaptive Immunity Group in Masaryk University, Brno and Genomics of Adaptive Immunity Lab in Institute of Bioorganic Chemistry, Moscow). Sample preparation and sequencing was performed by Dr. Olga Britanova and Dr. Maria Turchaninova. Raw sequencing reads were pre-processed and uploaded by Dr. Mikhail Shugay.

Supplementary material 2 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Figure S2

Supplementary material 5 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Table S3

Supplementary material 6 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Table S4

Supplementary material 1 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Figure S1

Supplementary material 4 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Table S2

Supplementary material 3 from: Reid BN, Servis JA, Timmers M, Rohwer F, Naro-Maciel E (2022) 18S rDNA amplicon sequence data (V1–V3) of the Palmyra Atoll National Wildlife Refuge, Central Pacific. Metabarcoding and Metagenomics 6: e78762. https://doi.org/10.3897/mbmg.6.78762

Table S1

opencc-by-4.0Nov 2024View details →

Rapid multiplexed nanopore amplicon sequencing to distinguish Plasmodium falciparum recrudescence from new infection in antimalarial drug trials

Open the record for dataset details and reuse information.

opencc-zeroJun 2022View details →

Supplementary material 2 from: Naro-Maciel E, Ingala MR, Werner IE, Reid BN, Fitzgerald AM (2022) COI amplicon sequence data of environmental DNA collected from the Bronx River Estuary, New York City. Metabarcoding and Metagenomics 6: e80139. https://doi.org/10.3897/mbmg.6.80139

Tables S1,S2, Figures S1–S3

opencc-zeroJun 2022View details →

Supplementary material 1 from: Naro-Maciel E, Ingala MR, Werner IE, Reid BN, Fitzgerald AM (2022) COI amplicon sequence data of environmental DNA collected from the Bronx River Estuary, New York City. Metabarcoding and Metagenomics 6: e80139. https://doi.org/10.3897/mbmg.6.80139

Supplementary Data Files 1, 2