An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies
Research article published in GigaScience (2019)
Abstract
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Резюме
The use of environmental DNA for species detection via metabarcoding is growing rapidly.
Почему это важно для гирудотерапии
В данном исследовании представлен лабораторный протокол и биоинформатический пайплайн для метабаркодинга экологической ДНК, разработанный для минимизации контаминации образцов и ошибок таксономического отнесения за счёт технической репликации, мультимаркерного подхода (12S, 16S, CytB), ПЦР с двойным мечением (twin-tagging PCR) и вероятностного таксономического отнесения с помощью программы PROTAX. Пайплайн был протестирован на ДНК, полученной из беспозвоночных, — 1532 пиявок из Сабаха (Малайзия), — что позволило выявить 162 детекции позвоночных при строгих критериях (из 95 образцов пиявок) и 190 — при менее строгих критериях (из 109 образцов пиявок). В данном исследовании пиявки выступают в роли пассивных коллекторов ДНК позвоночных для целей экологического и природоохранного мониторинга, а не как объекты терапевтических исследований или исследований секретома. Эта работа не имеет прямого отношения к гирудотерапии или области интересов ASH.
Цитирование
An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies
Axtner J et al. · GigaScience, 2019
Связанный клинический контекст
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