Malhi, R. | bioRxiv Science

bioRxiv2022

Non-invasive fecal DNA yields whole genome and metagenomic data for species conservation

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, and/or dangerous species. Integrating genomic techniques that use non-invasive biological samples with advances in computational approaches can benefit and inform wildlife conservation and management. Here we present a molecular pipeline that uses non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at 6X coverage) and metagenomic sequences, combining in a novel fashion widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome and/or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated by this pipeline was demonstrated by the application of genome-scale analysis and metagenomics to zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses generated compositional taxon classifications that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of our approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular pipeline presented here extends applications beyond what has previously been shown for target-enriched datasets and contributes towards the expansion and application of genomic techniques to conservation science and practice.

genomics↗

bioRxiv2022

NUMT PARSER: automated identification and removal of nuclear mitochondrial pseudogenes (numts) for accurate mitochondrial genome reconstruction in Panthera

Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from two ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW to two other bioinformatic approaches that can be used to account for numt contamination. We found that NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. NO_SCPLOWUMTC_SCPLOW PO_SCPLOWARSERC_SCPLOW therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.

evolutionary biology↗