Semi-quantitative characterisation of mixed pollen samples using MinION sequencing and Reverse Metagenomics (RevMet)
Semi-quantitative characterisation of mixed pollen samples using MinION sequencing and Reverse Metagenomics (RevMet)
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DOI：
10.1111/2041-210x.13265
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发表时间：
2019-10-01
影响因子：
6.6
通讯作者：
Yu, Douglas W.
中科院分区：
文献类型：
--
作者：
Peel, Ned;Dicks, Lynn V.;Yu, Douglas W.
鉴定和量化构成花粉混合物种样本的组成植物物种的能力在生态学、保护和农业中具有重要的应用。近年来，人们开发了花粉元条形码技术，以识别花粉的组成植物物种，但人们有充分的理由怀疑元条形码技术能否准确地量化花粉的相对丰度。无pcr的散弹枪宏基因组学方法在准确定量物种相对丰度方面具有更大的潜力，但由于参考基因组数量较少，将宏基因组学应用于真核生物具有挑战性。我们已经开发了RevMet（反向宏基因组学）管道，可以在不需要参考基因组的情况下，对混合物种真核生物样本（如蜜蜂采集的花粉）的物种组成进行可靠和半定量的表征。相反，参考物种只能用“基因组略图”来表示：低成本、低覆盖率、短读序列数据集。使用MinION（一种便携式纳米孔测序设备）从混合物种样本中对这些皮肤进行了单独的长reads测序，每个长reads都被唯一地分配给一个植物物种。我们对49种野生英国植物进行了基因组扫描，用已知成分的模拟DNA混合物验证了我们的管道，然后将RevMet应用于从野生蜜蜂收集的花粉负荷。我们证明RevMet可以在DNA bb0 = 1%的比例下识别混合物种样本中的植物物种，很少有假阳性和假阴性，并且可以可靠地区分样品中DNA含量高和含量低的物种。RevMet可以很容易地用于生成半定量数据集，用于广泛的混合真核生物样品。我们每个样本的成本是每个基因组脱脂90英镑，每个花粉样本60英镑，现在可用的新版本测序仪将进一步降低这些成本。
The ability to identify and quantify the constituent plant species that make up a mixed-species sample of pollen has important applications in ecology, conservation, and agriculture. Recently, metabarcoding protocols have been developed for pollen that can identify constituent plant species, but there are strong reasons to doubt that metabarcoding can accurately quantify their relative abundances. A PCR-free, shotgun metagenomics approach has greater potential for accurately quantifying species relative abundances, but applying metagenomics to eukaryotes is challenging due to low numbers of reference genomes. We have developed a pipeline, RevMet (Reverse Metagenomics) that allows reliable and semi-quantitative characterization of the species composition of mixed-species eukaryote samples, such as bee-collected pollen, without requiring reference genomes. Instead, reference species are represented only by 'genome skims': low-cost, low-coverage, short-read sequence datasets. The skims are mapped to individual long reads sequenced from mixed-species samples using the MinION, a portable nanopore sequencing device, and each long read is uniquely assigned to a plant species. We genome-skimmed 49 wild UK plant species, validated our pipeline with mock DNA mixtures of known composition, and then applied RevMet to pollen loads collected from wild bees. We demonstrate that RevMet can identify plant species present in mixed-species samples at proportions of DNA >= 1%, with few false positives and false negatives, and reliably differentiate species represented by high versus low amounts of DNA in a sample. RevMet could readily be adapted to generate semi-quantitative datasets for a wide range of mixed eukaryote samples. Our per-sample costs were 90 pound per genome skim and 60 pound per pollen sample, and new versions of sequencers available now will further reduce these costs.