Data from: Comparing the effectiveness of metagenomics and metabarcoding for diet analysis of a leaf-feeding monkey (Pygathrix nemaeus)

Fecal samples are of great value as a non-invasive means to gather information on the genetics, distribution, demography, diet, and parasite infestation of endangered species. Direct shotgun sequencing of fecal DNA could give information on these simultaneously, but this approach is largely untested. Here we use two fecal samples to characterize the diet of two Red-Shanked Doucs Langurs (Pygathrix nemaeus) that were fed a known combination of foliage, fruits, vegetables and cereals. Illumina HiSeq sequencing produced ~70 million paired reads per sample, of which ~10000 (0.014%) and ~44000 (0.066%) respectively corresponded to chloroplast genomes. Sequences were matched against a database of available chloroplast 'barcodes' for angiosperms. The results were compared with 'metabarcoding' using PCR amplification of the P6 loop of trnL. Shotgun sequencing identified 7 and 9 of the likely 16 diet plants, against 6 and 5 plant species identified by metabarcoding. Metabarcoding produced thousands of reads that were consistent with the known diet, but the barcodes were too short to identify several diet plants to genus. Metagenomics could utilize multiple, longer barcodes that combined had greater power of identification, but rare diet items were not recovered. Read numbers for diet species in metagenomic and metabarcoding data were correlated, indicating that both approaches are useful for determining relative sequence abundance. Metagenomic reads were uniformly distributed across the chloroplast genomes; thus if chloroplast genomes were to be used as reference, the precision of identifications and species recovery would improve further. Metagenomics also recovered the host mitochondrial genome and numerous intestinal parasite sequences in addition to generating data useful for characterizing the microbiome.

粪便样本作为非侵入性采样手段，可用于获取濒危物种的遗传学特征、分布格局、种群统计信息、饮食组成及寄生虫感染状况相关数据，具有极高的研究与应用价值。直接对粪便DNA进行鸟枪法测序（shotgun sequencing）可同时获取上述各类信息，但该方法目前尚未得到充分验证。本研究采用两份粪便样本，对投喂已知配比枝叶、果实、蔬菜与谷物的两只红腿白臀叶猴（Pygathrix nemaeus）的饮食组成进行解析。依托Illumina HiSeq测序平台，每份样本产出约7000万条双端读段（paired reads），其中分别有约10000条（占比0.014%）与44000条（占比0.066%）的读段匹配叶绿体基因组（chloroplast genome）。将所得序列与公开的被子植物（angiosperms）叶绿体条形码（chloroplast barcode）数据库进行比对。将该结果与基于trnL基因P6环的聚合酶链式反应（PCR）扩增的元条形码测序（metabarcoding）结果进行对比分析。鸟枪法测序成功鉴定出16种预期膳食植物中的7种与9种，而元条形码测序仅鉴定出6种与5种植物物种。元条形码测序可产出数千条与已知饮食组成匹配的读段，但由于其条形码序列过短，无法将部分膳食植物鉴定至属级水平。宏基因组学（metagenomics）可依托多条更长的条形码序列，组合后具备更强的物种鉴定能力，但无法检出丰度较低的膳食组分。宏基因组学与元条形码测序数据中，膳食物种的读段数量呈显著相关，表明两种方法均可用于推断序列的相对丰度。宏基因组学的读段在叶绿体基因组上分布均匀，因此若以叶绿体基因组作为参考序列，物种鉴定的准确性与物种检出率将进一步提升。此外，宏基因组学不仅可生成用于表征微生物组的有效数据，还能同时获取宿主的线粒体基因组以及大量肠道寄生虫序列。