Identification of known and novel recurrent viral sequences in data from multiple patients and multiple cancers

Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. We applied the workflow to 686 sequencing libraries from 252 cancer samples of different cancer and tissue types, 32 non-template controls, and 24 test samples. Recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. We provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. Unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified

Calculation of Tajima’s D and other neutrality test statistics from low depth next-generation sequencing data

BACKGROUND: A number of different statistics are used for detecting natural selection using DNA sequencing data, including statistics that are summaries of the frequency spectrum, such as Tajima’s D. These statistics are now often being applied in the analysis of Next Generation Sequencing (NGS) data. However, estimates of frequency spectra from NGS data are strongly affected by low sequencing coverage; the inherent technology dependent variation in sequencing depth causes systematic differences in the value of the statistic among genomic regions. RESULTS: We have developed an approach that accommodates the uncertainty of the data when calculating site frequency based neutrality test statistics. A salient feature of this approach is that it implicitly solves the problems of varying sequencing depth, missing data and avoids the need to infer variable sites for the analysis and thereby avoids ascertainment problems introduced by a SNP discovery process. CONCLUSION: Using an empirical Bayes approach for fast computations, we show that this method produces results for low-coverage NGS data comparable to those achieved when the genotypes are known without uncertainty. We also validate the method in an analysis of data from the 1000 genomes project. The method is implemented in a fast framework which enables researchers to perform these neutrality tests on a genome-wide scale

Treated like dirt: Robust forensic and ecological inferences from soil eDNA after challenging sample storage

Abstract Biodiversity of soil is routinely assessed with environmental DNA—most often by massive parallel sequencing of marker genes (eDNA metabarcoding). Soil biodiversity may be investigated in relation to biodiversity research or as a tool in forensic investigations. After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes, it is desirable to reduce biological activity, e.g., by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to an item of interest for protracted periods of time. Here, we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions and contrasting exposure (open/closed tubes). We used four different primer sets targeting bacteria, fungi, protists (cercozoans), and general eukaryotes. We quantified differences in richness, evenness, and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset. We found stronger community composition differences with extended storage time and with higher storage temperature, and differences between open and closed tubes. However, for samples stored <28 days at a maximum of 20°C, changes were generally insignificant. Classification models successfully assigned most samples to their exact location of origin and correct habitat type even after 480 days storage. Even samples showing larger changes generally retained the original sample as the best match. For most biodiversity and forensic applications, storage of samples for days and even several weeks may thus not be a problem, if storage temperature does not exceed 20°C

Systematics of West African Miniopterus with the description of a new species

The phylogenetic relationships and species limits within the chiropteran family Miniopteridae are poorly known in mainland Africa. Recent systematic studies in Madagascar have shown that this is a species-rich family, yet only eight species are currently recognized or hypothesized for continental Africa. Based on partial cytochrome b sequences and morphometric analysis, we describe a new species of Miniopterus that is endemic to a restricted, montane region of Liberia and Guinea. Furthermore, the taxonomic status of the West African Miniopterus schreibersii villiersi is resolved and shown to be a distinct species, M. villiersi. that is not closely related to M. schreibersii. Finally, the species M. inflatus is revealed to be paraphyletic, with the central African rainforest populations apparently not closely related to the savanna forms in eastern and southern Africa. Based on the results of this study. the number of Miniopterus species in Africa has increased from eight to 11, with more cryptic species likely to be discovered

Miniopterus nimbae Monadjem & S Hapiro & Richards & Karabulut & Crawley & Nielsen & Hansen & Bohmann & Mourier 2019, sp. nov.

&lt;i&gt;Miniopterus nimbae&lt;/i&gt; sp. nov. &lt;p&gt;Nimba long-fingered bat&lt;/p&gt; &lt;p&gt; &lt;i&gt;Holotype&lt;/i&gt;&lt;/p&gt; &lt;p&gt;DM 12621 (field no. AM2010_12_18_1), an adult male, was collected by Ara Monadjem. The specimen was fixed in formalin and then transferred to 70% alcohol. The skull has been extracted and cleaned. Photograph of the skull and drawing of the tragus of the holotype are illustrated in Figs. 6 and 7.&lt;/p&gt; &lt;p&gt; &lt;i&gt;Type locality&lt;/i&gt;&lt;/p&gt; &lt;p&gt;Liberia, Nimba Province, Mount Gangra, 10 km to the west of Mount Nimba (Fig. 1). The bat was netted on 18 December 2010 exiting from a mine adit mid-way up Mount Gangra (7.55434&deg;N, 8.62902&deg;W) at 720 m a.s.l, in secondary forest.&lt;/p&gt; &lt;p&gt; &lt;i&gt;Paratypes&lt;/i&gt;&lt;/p&gt; &lt;p&gt;No other specimens of this species were captured or collected on the same day at the same site. However, the previous night an adult female (DM 12614) was collected at the Yiti River 9 km to the south-east of Mount Gangra. Photograph of the paratype is illustrated in Fig. 8.&lt;/p&gt; &lt;p&gt; &lt;i&gt;Etymology&lt;/i&gt;&lt;/p&gt; &lt;p&gt; This species is named after Mount Nimba, one of just three localities from which it is known, further highlighting the critical importance of this mountain for bat conservation in Africa (Monadjem &lt;i&gt;et al&lt;/i&gt;., 2016).&lt;/p&gt; &lt;p&gt; &lt;i&gt;Diagnosis&lt;/i&gt;&lt;/p&gt; &lt;p&gt; This is a large-sized &lt;i&gt;Miniopterus&lt;/i&gt; from Mount Nimba, Liberia, with a mean forearm length of 47.4 mm (&lt;i&gt;n&lt;/i&gt; = 26 individuals). The large size of this bat (particularly its forearm length) readily distinguishes &lt;i&gt;M. nimbae&lt;/i&gt; from all other African &lt;i&gt;Miniopterus&lt;/i&gt; taxa except the &lt;i&gt;M. inflatus&lt;/i&gt; / &lt;i&gt;M. africanus&lt;/i&gt; group. In external and craniodental measurements, &lt;i&gt;M. nimbae&lt;/i&gt; is similar in size to other members of the &lt;i&gt;M. inflatus&lt;/i&gt; group (&lt;i&gt;M. inflatus&lt;/i&gt; s.s., &lt;i&gt;M&lt;/i&gt;. cf. &lt;i&gt;inflatus&lt;/i&gt; and &lt;i&gt;M. africanus&lt;/i&gt;) (Tables 3&ndash;5); however, in multi-dimensional morphospace based on craniodental measurements, it overlaps only with &lt;i&gt;M. inflatus&lt;/i&gt; s.s. The taxon &lt;i&gt;M&lt;/i&gt;. cf. &lt;i&gt;inflatus&lt;/i&gt; from eastern and southern Africa tends to have a light pelage, being more reddish-brown in colour (compared with a deep chocolate brown in &lt;i&gt;M. nimbae&lt;/i&gt;).&lt;/p&gt; &lt;p&gt; It is not possible, at present to distinguish &lt;i&gt;M. nimbae&lt;/i&gt; from &lt;i&gt;M. inflatus&lt;/i&gt; s.s. on external characters. However, they can be readily distinguished by cranial features. In particular, the 1st upper premolar has an additional lingual cusp, posterior to the main cusp, that is present in &lt;i&gt;M. nimbae&lt;/i&gt; but absent in &lt;i&gt;M. inflatus&lt;/i&gt; s.s. (Fig. 9); this feature being clearly visible, even with a low magnification hand lens, and consistently present in all the specimens examined in this study. The lower tooth row (i 1 &ndash;m 3) and lower molar (LWMOLS) lengths are also slightly larger in &lt;i&gt;M. nimbae&lt;/i&gt; than &lt;i&gt;M. inflatus&lt;/i&gt; s.s. In terms of cranial geometry, &lt;i&gt;M. nimbae&lt;/i&gt; differs from &lt;i&gt;M. inflatus&lt;/i&gt; s.s. as it bears a slightly more gracile skull, less &lsquo;inflated&rsquo; braincase, and the point of maximum curvature along the occiput is more elevated in the newly described taxon than in the nominate form. Furthermore, these two taxa, are also distinguishable on molecular grounds (K2P pairwise genetic distance = 1.6%). Additionally, the ranges of the two taxa do not appear to overlap. &lt;i&gt;Miniopterus africanus&lt;/i&gt; appears to be restricted to north-eastern Africa and is genetically distinct (K2P pairwise distance = 16.7%) from &lt;i&gt;M. nimbae&lt;/i&gt;.&lt;/p&gt; &lt;p&gt; &lt;i&gt;Description&lt;/i&gt;&lt;/p&gt; &lt;p&gt; External characters.&mdash; &lt;i&gt;Miniopterus nimbae&lt;/i&gt; is large-sized for the genus, but showing typical generic features including a rounded head, an elongated second phalanx of the third digit, rounded ears, and a relatively long and straight tragus. The tail is slightly less than half that of the total length. The pelage is dark chocolate brown above and slightly paler below. Individual hairs are unicoloured. The mass and standard external measurements of the holotype compared with a small sample of other individuals are shown in Table 3.&lt;/p&gt; &lt;p&gt; Craniodental characters.&mdash;The skull is robust for a &lt;i&gt;Miniopterus&lt;/i&gt; species. The rostrum is broad and the braincase is rounded and high, typical for the genus of &lt;i&gt;Miniopterus&lt;/i&gt;. The dentition of &lt;i&gt;M. nimbae&lt;/i&gt; is I 2/3, C 1/1, P 2/3, M 3/3, which is typical of the genus. In the upper tooth row, the inner incisor is larger than the outer one, and the anterior premolar is relatively well developed. The cranial and dental measurements of the holotype compared with a small sample of other individuals are shown in Tables 4 and 5.&lt;/p&gt; &lt;p&gt; &lt;i&gt;Distribution&lt;/i&gt;&lt;/p&gt; &lt;p&gt; Analysis of genetic samples taken from five specimens with large forearm lengths (range 46.9&ndash; 48.2 mm) at Mount Gangra (Nimba, Liberia), group them all as &lt;i&gt;M. nimbae&lt;/i&gt; (Figs. 2 and 3). Although not genetically analysed, it is likely that specimens previously collected from northern Liberia or southeastern Guinea and identified as &lt;i&gt;M. inflatus&lt;/i&gt; are instead referable to this new species, &lt;i&gt;M. nimbae&lt;/i&gt;. Based on this assumption, this taxon is known from just three localities: Mount Nimba (and surrounding uplands), Liberia; Wonegizi Mountains, Liberia; and Mount B&eacute;ro, Guinea (Wolton &lt;i&gt;et al&lt;/i&gt;., 1982; Koopman &lt;i&gt;et al&lt;/i&gt;., 1995; Fahr &lt;i&gt;et al&lt;/i&gt;., 2006; Monadjem &lt;i&gt;et al&lt;/i&gt;., 2016). The closest locality for &lt;i&gt;M. inflatus&lt;/i&gt; s.s. is at least 2,000 km away in eastern Nigeria (Happold, 2013 &lt;i&gt;b&lt;/i&gt;), and no other large &lt;i&gt;Miniopterus&lt;/i&gt; specimens have been collected in the intermediate area, despite extensive surveys in a number of localities e.g., Tha&iuml; and Como&eacute; National Parks in C&ocirc;te d&rsquo;Ivoire (Fahr and Kalko, 2011), the Simandou Range in eastern Guinea (Decher &lt;i&gt;et al&lt;/i&gt;., 2015), the Fouta Djallon mountains in central Guinea (Weber and Fahr, 2007) or anywhere in Ghana or Sierra Leone (Grubb &lt;i&gt;et al&lt;/i&gt;., 1998; Happold, 2013 &lt;i&gt;b&lt;/i&gt;, 2013 &lt;i&gt;c&lt;/i&gt;). Therefore, &lt;i&gt;M. nimbae&lt;/i&gt; is probably an endemic to the upland areas of northern Liberia and south-eastern Guinea, and may be shown to also occur in the upland area of western C&ocirc;te d&rsquo;Ivoire at and around Mount Nimba.&lt;/p&gt; &lt;p&gt; Biology: Practically nothing is known about the biology of this Upper Guinea forest endemic. It has been recorded roosting in mine adits at 720&ndash; 970 m above sea level (a.s.l.) at Mount Gangra and Mount Yuelliton (both within 10 km of Mount Nimba). The size of one roosting colony at Mount Gangra was estimated at between 20&ndash;30 individuals; the site was shared with large numbers of &lt;i&gt;Myonycteris angolensis&lt;/i&gt; and &lt;i&gt;Hipposideros&lt;/i&gt; cf. &lt;i&gt;ruber&lt;/i&gt;, and a few &lt;i&gt;H. marisae&lt;/i&gt;. A second roosting colony was estimated to include&gt; 200 individuals of this species at Mount Yuelliton (which was not inhabited by any other bat species &mdash; A. Monadjem, personal observation). It has been netted at various localities in the foothills of Mount Nimba and in the low-lying rainforest region between these three upland areas. This suggests that this species roosts in upland areas (700 m a.s.l.), and then descends to forage in lower lying forested areas (about 500 m a.s.l.). In a sample of 13 females captured at Nimba between late December and end-March, five were pregnant. During the same period, three out of 10 males had scrotal testes. The mean frequency of the knee of handreleased &lt;i&gt;M. nimbae&lt;/i&gt; captured at Mount Nimba was 48.4 kHz (range: 47.2&ndash;48.9 kHz, &lt;i&gt;n&lt;/i&gt; = 4).&lt;/p&gt; &lt;p&gt; &lt;i&gt;Other Taxonomic Considerations&lt;/i&gt;&lt;/p&gt; &lt;p&gt; In addition to the description of the new species, &lt;i&gt;M. nimbae&lt;/i&gt;, the phylogeny presented here (Figs. 2 and 3) also identifies two other distinct taxa. The first is the taxon &lt;i&gt;M. villiersi&lt;/i&gt;, which does not appear to be closely related to &lt;i&gt;M. schreibersii&lt;/i&gt; s.s. and should therefore not be considered a subspecies of the latter mentioned taxon. In fact, &lt;i&gt;M. villiersi&lt;/i&gt; is sister to &lt;i&gt;M. nimbae&lt;/i&gt; (Figs. 2 and 3), from which it is readily distinguishable based on genetics and size (see Tables 2&ndash;5); the pairwise genetic distance between the two species is 9.0%, and there is no overlap between these two species in forearm length or any of the craniodental measurements presented here. The echolocation calls also differ, with the frequency of the knee of &lt;i&gt;M. villiersi&lt;/i&gt; calls (based on hand-released individuals that were captured at Mount Nimba &mdash; &lt;i&gt;x&lt;/i&gt; = 51.6 kHz, range 51.1&ndash;52.4 kHz, &lt;i&gt;n&lt;/i&gt; = 5) being distinctly higher than that of &lt;i&gt;M. nimbae&lt;/i&gt; (&lt;i&gt;x&lt;/i&gt; = 48.4 kHz, range 47.2&ndash;48.9 kHz, &lt;i&gt;n&lt;/i&gt; = 4) with no overlap between the two species.&lt;/p&gt; &lt;p&gt; The second taxon refers to the &lt;i&gt;M. inflatus&lt;/i&gt; group, which appears to be paraphyletic. &lt;i&gt;Miniopterus inflatus&lt;/i&gt; s.s. (based on sequenced specimens from Gabon, and close to the type locality in southern Cameroon) is sister to the taxon &lt;i&gt;M. nimbae&lt;/i&gt; and these two are sister to &lt;i&gt;M. villiersi&lt;/i&gt; (Figs. 2 and 3). By contrast, the &lt;i&gt;M&lt;/i&gt;. cf. &lt;i&gt;inflatus&lt;/i&gt; specimens (from Malawi and Mozambique) are sister to &lt;i&gt;M. fraterculus&lt;/i&gt; and &lt;i&gt;M. minor&lt;/i&gt;. This suggests that the taxon &lt;i&gt;M. inflatus&lt;/i&gt; s.l. comprises, in addition to the newly recognised &lt;i&gt;M. nimbae&lt;/i&gt;, two distinct and not closely related taxa which we refer to as &lt;i&gt;M. inflatus&lt;/i&gt; s.s. (from Gabon), and &lt;i&gt;M&lt;/i&gt;. cf. &lt;i&gt;inflatus&lt;/i&gt; (from Malawi and Mozambique).&lt;/p&gt;Published as part of &lt;i&gt;Monadjem, Ara, Shapiro, Julie T., Richards, Leigh R., Karabulut, Hatice, Crawley, Wing, Nielsen, Ida Broman, Hansen, Anders, Bohmann, Kristine &amp; Mourier, Tobias, 2019, Systematics of West African Miniopterus with the description of a new species, pp. 237-256 in Acta Chiropterologica 21 (2)&lt;/i&gt; on pages 245-248, DOI: 10.3161/15081109ACC2019.21.2.001, &lt;a href="http://zenodo.org/record/3944920"&gt;http://zenodo.org/record/3944920&lt;/a&gt