Genome‐skimming provides accurate quantification for pollen mixtures

Lang, Dandan; Tang, Min; Hu, Jiahui; Zhou, Xin

doi:10.1111/1755-0998.13061

Cited by 34 publications

(40 citation statements)

References 72 publications

(94 reference statements)

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“…If genome skimming is a viable option (there is enough DNA present), closely related species like Echinacea can be differentiated [43]. Additionally, there is support for the fact that genome skims of plants can also be at least semiquantitative [44]. This assertation is not without speculation since complications such as degradation of DNA, genome size, and if chloroplast genes are used, as they were here, the number of plastids per cell can vary among plant tissues for a number of reasons [45].…”

Section: Original Papersmentioning

confidence: 99%

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea

et al. 2021

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The use of DNA-based methods to authenticate botanical dietary supplements has been vigorously debated for a variety of reasons. More comparisons of DNA-based and chemical methods are needed, and concordant evaluation of orthogonal approaches on the same products will provide data to better understand the strengths and weaknesses of both approaches. The overall application of DNA-based methods is already firmly integrated into a wide array of continually modernizing stand alone and complementary authentication protocols. Recently, the use of full-length chloroplast genome sequences provided enhanced discriminatory capacity for closely related species of Echinacea compared to traditional DNA barcoding approaches (matK and rbcL). Here, two next-generation sequencing approaches were used: (1) genome skimming and (2) PCR amplicon (metabarcoding). The two genetic approaches were then combined with HPLC-UV to evaluate 20 commercially available dietary supplements of Echinacea representing “finished” products. The trade-offs involved in different DNA approaches were discussed, with a focus on how DNA methods support existing, accepted chemical methods. In most of the products (19/20), HPLC-UV suggested the presence of Echinacea spp. While metabarcoding was not useful with this genus and instead only resolved 7 products to the family level, genome skimming was able to resolve to species (9) or genus (1) with the 10/20 products where it was successful. Additional ingredients that HPLC-UV was unable to identify were also found in four products along with the relative sequence proportion of the constituents. Additionally, genome skimming was able to identify one product that was a different Echinacea species entirely.

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Section: Original Papersmentioning

confidence: 99%

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea

et al. 2021

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“…Genetic and genomic data are of critical importance for many applications, including species delimitation [1][2][3], studies on evolution and phylogenies [4][5][6], biodiversity assessments and conservation [7,8], reconstructions of past plant communities [9][10][11], or for more applied tasks such as forensics [12,13], pollination and food web studies [14][15][16] and monitoring of invasive species [17]. While many of these tasks can be undertaken by sequencing plastid or rDNA amplicons [1,2,18,19], increasing emphasis has been given to the potential of using genomic data for DNA barcoding and wider phylogenomic studies [4,[20][21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

The Treasure Vault Can be Opened: Large-Scale Genome Skimming Works Well Using Herbarium and Silica Gel Dried Material

Alsos

Lavergne

Merkel

et al. 2020

Plants

104

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Genome skimming has the potential for generating large data sets for DNA barcoding and wider biodiversity genomic studies, particularly via the assembly and annotation of full chloroplast (cpDNA) and nuclear ribosomal DNA (nrDNA) sequences. We compare the success of genome skims of 2051 herbarium specimens from Norway/Polar regions with 4604 freshly collected, silica gel dried specimens mainly from the European Alps and the Carpathians. Overall, we were able to assemble the full chloroplast genome for 67% of the samples and the full nrDNA cluster for 86%. Average insert length, cover and full cpDNA and rDNA assembly were considerably higher for silica gel dried than herbarium-preserved material. However, complete plastid genomes were still assembled for 54% of herbarium samples compared to 70% of silica dried samples. Moreover, there was comparable recovery of coding genes from both tissue sources (121 for silica gel dried and 118 for herbarium material) and only minor differences in assembly success of standard barcodes between silica dried (89% ITS2, 96% matK and rbcL) and herbarium material (87% ITS2, 98% matK and rbcL). The success rate was > 90% for all three markers in 1034 of 1036 genera in 160 families, and only Boraginaceae worked poorly, with 7 genera failing. Our study shows that large-scale genome skims are feasible and work well across most of the land plant families and genera we tested, independently of material type. It is therefore an efficient method for increasing the availability of plant biodiversity genomic data to support a multitude of downstream applications.

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“…2019). Fortunately, methods for estimating within-sample species frequencies are being developed in metagenomic pipelines (Lang et al . 2019; Peel et al .…”

Section: Discussionmentioning

confidence: 99%

Biodiversity Soup II: A bulk-sample metabarcoding pipeline emphasizing error reduction

Yang

Bohmann

Wang

et al. 2020

Preprint

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AbstractDespite widespread recognition of its great promise to aid decision-making in environmental management, the applied use of metabarcoding requires improvements to reduce the multiple errors that arise during PCR amplification, sequencing, and library generation. We present a co-designed wet-lab and bioinformatic workflow for metabarcoding bulk samples that removes both false-positive (tag jumps, chimeras, erroneous sequences) and false-negative (‘drop-out’) errors. However, we find that it is not possible to recover relative-abundance information from amplicon data, due to persistent species-specific biases.To present and validate our workflow, we created eight mock arthropod soups, all containing the same 248 arthropod morphospecies but differing in absolute and relative DNA concentrations, and we ran them under five different PCR conditions. Our pipeline includes qPCR-optimized PCR annealing temperature and cycle number, twin-tagging, multiple independent PCR replicates per sample, and negative and positive controls. In the bioinformatic portion, we introduce Begum, which is a new version of DAMe (Zepeda Mendoza et al. 2016. BMC Res. Notes 9:255) that ignores heterogeneity spacers, allows primer mismatches when demultiplexing samples, and is more efficient. Like DAMe, Begum removes tag-jumped reads and removes sequence errors by keeping only sequences that appear in more than one PCR at above a minimum copy number.We report that OTU dropout frequency and taxonomic amplification bias are both reduced by using a PCR annealing temperature and cycle number on the low ends of the ranges currently used for the Leray-Fol-Degen-Rev primers. We also report that tag jumps and erroneous sequences can be nearly eliminated with Begum filtering, at the cost of only a small rise in drop-outs. We replicate published findings that uneven size distribution of input biomasses leads to greater drop-out frequency and that OTU size is a poor predictor of species input biomass. Finally, we find no evidence that different primer tags bias PCR amplification (‘tag bias’).To aid learning, reproducibility, and the design and testing of alternative metabarcoding pipelines, we provide our Illumina and input-species sequence datasets, scripts, a spreadsheet for designing primer tags, and a tutorial.

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Genome‐skimming provides accurate quantification for pollen mixtures

Cited by 34 publications

References 72 publications

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea

The Treasure Vault Can be Opened: Large-Scale Genome Skimming Works Well Using Herbarium and Silica Gel Dried Material

Biodiversity Soup II: A bulk-sample metabarcoding pipeline emphasizing error reduction

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