An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies

	An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies
	Axtner, J; Crampton-Platt, A; Horig, LA; Mohamed, A; Xu, CCY; Yu, DW; Wilting, A
	2019
发表期刊	GIGASCIENCE
ISSN	2047-217X
卷号	8 期号:4 页码:-
摘要	Background: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. Findings: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S, 16S, CytB); (iii) a twin-tagging, 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. Conclusions: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.
收录类别	SCI
语种	英语
文献类型	期刊论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/12990
专题	科研部门_生态学与环境保护中心（俞维理）
推荐引用方式 GB/T 7714	Axtner, J,Crampton-Platt, A,Horig, LA,et al. An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies[J]. GIGASCIENCE,2019,8(4):-.
APA	Axtner, J.,Crampton-Platt, A.,Horig, LA.,Mohamed, A.,Xu, CCY.,...&Wilting, A.(2019).An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies.GIGASCIENCE,8(4),-.
MLA	Axtner, J,et al."An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies".GIGASCIENCE 8.4(2019):-.

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