Metatranscriptomics yields new genomic resources and sensitive detection of infections for diverse blood parasites

Metatranscriptomics is a powerful method for studying the composition and function of complex microbial communities. The application of metatranscriptomics to multi-species parasite infections is of particular interest, as research on parasite evolution and diversification has been hampered by technical challenges to genome-scale DNA sequencing. In particular, blood parasites of vertebrates are abundant and diverse though they often occur at low infection intensities and exist as multi-species infections, rendering the isolation of genomic sequence data challenging. Here, we use birds and their diverse haemosporidian parasites to illustrate the potential for metatranscriptome sequencing to generate large quantities of genome-wide sequence data from multiple blood parasite species simultaneously. We used RNA-Seq on 24 blood samples from songbirds in North America to show that metatranscriptomes can yield large proportions of haemosporidian protein-coding gene repertoires even when infections are low-intensity (<0.1% red blood cells infected) and consist of multiple parasite taxa. By bioinformatically separating host and parasite transcripts and assigning them to the haemosporidian genus of origin, we found that transcriptomes detected ~23% more total parasite infections across all samples than were identified using microscopy and DNA barcoding. For single-species infections, we obtained data for upwards of 1,300 loci from samples with as low as 0.03% parasitemia, with the number of loci increasing with infection intensity. In total, we provide data for 1,502 single-copy orthologous loci from a phylogenetically-diverse set of 33 haemosporidian mitochondrial lineages. The metatranscriptomic approach described here has the potential to accelerate ecological and evolutionary research on haemosporidians and other diverse parasites.

宏转录组学（Metatranscriptomics）是解析复杂微生物群落组成与功能的有力研究手段。将宏转录组学应用于多物种寄生虫感染研究具有独特价值，此前寄生虫演化与多样化研究长期受限于基因组级DNA测序的技术瓶颈。脊椎动物血液寄生虫类群丰富、多样性极高，但这类寄生虫通常感染强度较低，且多以混合感染形式存在，这使得其基因组序列数据的分离获取极具挑战。本研究以鸟类及其多样的血孢子虫（haemosporidian）寄生虫为研究模型，证实宏转录组测序可同时从多种血液寄生虫物种中获取大规模全基因组序列数据。我们对北美鸣禽的24份血液样本开展RNA测序（RNA-Seq），结果显示：即便感染强度极低（感染红细胞占比<0.1%）且存在多寄生虫类群混合感染，宏转录组仍可获取血孢子虫蛋白编码基因库的大量组分。通过生物信息学方法分离宿主与寄生虫转录本，并将其归属至对应血孢子虫属，我们发现相较于显微镜检查与DNA条形码技术（DNA barcoding），宏转录组在所有样本中可多检出约23%的寄生虫感染总数。针对单物种感染样本，当寄生虫血症（parasitemia）低至0.03%时，我们仍可获取超过1300个基因座的相关数据，且基因座数量随感染强度提升而增加。本研究共计为33个系统发育多样性丰富的血孢子虫线粒体谱系提供了1502个单拷贝直系同源基因座（orthologous loci）的数据集。本文所阐述的宏转录组学方法，有望加速血孢子虫及其他多样寄生虫的生态学与演化研究进程。