Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.     

The Earth BioGenome project: opportunities and challenges for plant genomics and conservation

Sequencing them all. That is the ambitious goal of the recently launched Earth BioGenome project (Proceedings of the National Academy of Sciences of the United States of America, 115, 4325–4333), which aims to produce reference genomes for all eukaryotic species within the next decade. In this perspective, we discuss the opportunities of this project with a plant focus, but highlight also potential limitations. This includes the question of how to best capture all plant diversity, as the green taxon is one of the most complex clades in the tree of life, with over 300 000 species. For this, we highlight four key points: (i) the unique biological insights that could be gained from studying plants, (ii) their apparent underrepresentation in sequencing efforts given the number of threatened species, (iii) the necessity of phylogenomic methods that are aware of differences in genome complexity and quality, and (iv) the accounting for within‐species genetic diversity and the historical aspect of conservation genetics.     

Cryptosporidium species and subtypes in river water and riverbed sediment using next-generation sequencing

This study uncovered the prevalence, harboured species, and subtype diversity of Cryptosporidium species in river water and its sediment from the Apies River in South Africa. Cryptosporidium spp. concentrations in freshwater and its sediment were determined using Ziehl-Neelsen staining and quantitative Polymerase Chain Reaction (qPCR) techniques. Next-generation sequencing (NGS) targeting the 60 kDa glycoprotein (gp60) gene of Cryptosporidium spp. was performed to reveal the species, subtype families and subtypes harboured in freshwater and its sediment. Although the results revealed that water samples had a higher prevalence (30%) compared with sediment (28%), the number of observable Cryptosporidium spp. oocysts in sediment samples (ranging from 4.90 to 5.81 log₁₀ oocysts per 1 Liter) was higher than that of river water samples (ranging from 4.60 to 5.58 log₁₀ oocysts per 1 L) using Ziehl-Neelsen staining. The 18S ribosomal ribonucleic acid (rRNA) gene copy of Cryptosporidium in riverbed sediments ranged from 6.03 to 7.65 log₁₀, whereas in river water, it was found to be between 4.20 and 6.79 log₁₀. Subtyping results showed that in riverbed sediments, Cryptosporidium parvum accounted for 40.72% of sequences, followed by Cryptosporidium hominis with 23.64%, Cryptosporidium cuniculus with 7.10%, Cryptosporidium meleagridis with 4.44% and the least was Cryptosporidium wrairi with 2.59%. A considerable percentage of reads in riverbed sediment (21.25%) was not assigned to any subtype. River water samples had 45.63% of sequences assigned to C. parvum, followed by 30.32% to C. hominis, 17.99% to C. meleagridis and 5.88% to C. cuniculus. The data obtained are concerning, as Cryptosporidium spp. have intrinsic resistance to water treatment processes and low infectious doses, which can pose a risk to human health due to the various uses of water (for human consumption, leisure, and reuse).     

Genomic data reveal two distinct species from the widespread alpine ginger Roscoea tibetica Batalin (Zingiberaceae)

Species delimitation is a key foundation for exploring biodiversity. However, the existence of continuous phenotypic variation in widespread species challenges accurate species delimitation based on classical taxonomy. In this study, we investigated the cryptic diversity of a widespread herb (Roscoea tibetica Batalin) in a biodiversity hotspot (the Hengduan Mountains, China) using genotyping by sequencing, examining morphological traits, developing species distribution models, and simulating demographic history. Phylogenomic reconstruction, principal component analysis, and genetic structure inferences indicated that previously reported R. tibetica comprised two monophyletic lineages with a deep divergence. Several morphological diagnostic characteristics were discovered from field and common garden that corresponded to these independent evolutionary lineages. Species distribution models illustrated significant ecological divergence between both lineages. All evidence strongly supported that R. tibetica, as described in previous taxonomy, actually comprises two distinct species. Model test of gene flow and effective population size changes in fastsimcoal2, and a negative Tajima's D-value suggested that recent contact likely occurred between the two lineages. Our results proposed that cryptic diversity in previously reported R. tibetica was possibly associated with phenotypic plasticity in heterogeneous environments and morphological convergence in similar habitats. This study suggests that caution should be exercised when attempting to gain biological insight into species with large-scale morphological variation, and species delimitation should be done in advance.