Assessment of High Throughput Sequencing Quality of Host DNA Enriched From Faeces: A Case From Captive Tiger.

Faeces serve as a widely used non-invasive material in wildlife studies, from which host DNA can be enriched for high-throughput sequencing. However, the characteristics of faecal DNA sequencing, particularly the relationship between sequencing volume and genotyping accuracy or genomic coverage, have remained unclear. This knowledge gap makes it challenging to pre-estimate the required sequencing depth to ensure data quality while controlling costs. In this study, we used host-enriched faecal DNA obtained via peri-extraction enrichment by SDS (PEERS) to perform comparative analyses of whole-genome sequencing data from paired faecal and blood samples of 11 captive Amur tigers. We systematically evaluated how data volume affects genomic coverage and genotyping accuracy. Our results revealed that faecal sequencing reads cover the genome unevenly, which may compromise the reliability of underrepresented allele identification and subsequent genomic analyses. Specifically, genomic coverage and genotyping accuracy, including precision, error rate, recall and F1-score, improved with increasing sequencing depth in both blood and faecal DNA. For reliable genotyping (values > 0.95), a minimum average depth of 20× was required for blood-derived DNA, whereas faecal DNA needed up to 50× depth when the nuclear DNA enrichment efficiency (EE