Minimally destructive hDNA extraction method for retrospective genetics of pinned historical Lepidoptera specimens.

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies.

Molecular detection of Cryptosporidium in Alpine musk deer (Moschus chrysogaster) in Gansu Province, Northwest China.

Cryptosporidium spp. are protozoa commonly found in domestic and wild animals. Limited information is available on Cryptosporidium in deer worldwide. In this study, 201 fecal samples were collected from Alpine musk deer on three farms in Gansu Province, China. Detection and subtyping of Cryptosporidium were performed by PCR and sequence analysis of the SSU rRNA and gp60 genes. The prevalence of Cryptosporidium infection in Alpine musk deer was 3.9% (8/201), with infection rates of 1.0% (1/100), 2.8% (1/36), and 9.2% (6/65) in three different farms. All positive samples for Cryptosporidium were from adult deer. Two Cryptosporidium species were identified, including C. parvum (n = 2) and C. xiaoi (n = 6). The C. parvum isolates were subtyped as IIdA15G1, while the C. xiaoi isolates were subtyped as XXIIIa (n = 2) and XXIIIg (n = 4). The IIdA15G1 subtype of C. parvum was found for the first time in deer. These results provide important insights into the identity and human infectious potential of Cryptosporidium in farmed Alpine musk deer.

bsgenova: an accurate, robust, and fast genotype caller for bisulfite-sequencing data.

BACKGROUND: Bisulfite sequencing (BS-Seq) is a fundamental technique for characterizing DNA methylation profiles. Genotype calling from bisulfite-converted BS-Seq data allows allele-specific methylation analysis and the concurrent exploration of genetic and epigenetic profiles. Despite various methods have been proposed, single nucleotide polymorphisms (SNPs) calling from BS-Seq data, particularly for SNPs on chromosome X and in the presence of contaminative data, poses ongoing challenges. RESULTS: We introduce bsgenova, a novel SNP caller tailored for bisulfite sequencing data, employing a Bayesian multinomial model. The performance of bsgenova is assessed by comparing SNPs called from real-world BS-Seq data with those from corresponding whole-genome sequencing (WGS) data across three human cell lines. bsgenova is both sensitive and precise, especially for chromosome X, compared with three existing methods. Moreover, in the presence of low-quality reads, bsgenova outperforms other methods notably. In addition, bsgenova is meticulously implemented, leveraging matrix imputation and multi-process parallelization. Compared to existing methods, bsgenova stands out for its speed and efficiency in memory and disk usage. Furthermore, bsgenova integrates bsextractor, a methylation extractor, enhancing its flexibility and expanding its utility. CONCLUSIONS: We introduce bsgenova for SNP calling from bisulfite-sequencing data. The source code is available at https://github.com/hippo-yf/bsgenova under license GPL-3.0.

Genomic full-length sequence of the novel HLA-C*06:364 allele.

HLA-C*06:364 differs from HLA-C*06:02:01:01 by a non-synonymous nucleotide substitution in exon 3.

The novel KIR2DL3*037 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

The novel KIR2DL3*037 allele differs from the closest allele KIR2DL3*00101 by a single missense mutation.

A single nucleotide substitution in exon 5 generated the novel KIR2DL3*00112 allele.

The novel KIR2DL3*00112 allele differs from the closest allele KIR2DL3*00101 by a single same sense mutation.

Recognition of the HLA-DQB1*03:96 allele in a Taiwanese individual.

One nucleotide substitution in codon 130 of HLA-DQB1*03:03:02:01 results in a novel allele HLA-DQB1*03:96.

The novel HLA-A*02:1152 and HLA-B*40:566 alleles identified in Russian bone marrow donors.

Identification of two novel HLA alleles in Russian bone marrow donors.

Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies.

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is usually required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low read depth; (2) most of the benefit of short-read polishing occurs by 25× depth; (3) Polypolish-careful almost never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5×), Polypolish-careful and Pypolca-careful when depth is low (5-25×), and Polypolish-default and Pypolca-careful when depth is sufficient (>25×).

Eupransor demetentiae gen. nov., sp. nov., a novel fructophilic lactic acid bacterium from bumble bees.

Strain LMG 33000T was isolated from a Bombus lapidarius gut sample. It shared the highest percentage 16S rRNA sequence identity, average amino acid identity, and amino acid identity of conserved genes with Convivina intestini LMG 28291T (95.86 %, 69.9 and 76.2 %, respectively), and the highest percentage OrthoANIu value with Fructobacillus fructosus DSM 20349T (71.4 %). Phylogenomic analyses by means of 107 or 120 conserved genes consistently revealed Convivina as nearest neighbour genus. The draft genome of strain LMG 33000T was 1.44 Mbp in size and had a DNA G+C content of 46.1 mol%. Genomic and physiological analyses revealed that strain LMG 33000T was a typical obligately fructophilic lactic acid bacterium that lacked the adhE and aldh genes and that did not produce ethanol during glucose or fructose metabolism. In contrast, Convivina species have the adhE and aldh genes in their genomes and produced ethanol from glucose and fructose metabolism, which is typical for heterofermentative lactic acid bacteria. Moreover, strain LMG 33000T exhibited catalase activity, an unusual characteristic among lactic acid bacteria, that is not shared with Convivina species. Given its position in the phylogenomic trees, and the difference in genomic percentage G+C content and in physiological and metabolic characteristics between strain LMG 33000T and Convivina species, we considered it most appropriate to classify strain LMG 33000T into a novel genus and species within the Lactobacillaceae family for which we propose the name Eupransor demetentiae gen. nov., sp. nov., with LMG 33000T (=CECT 30958T) as the type strain.

Campylobacter devanensis sp. nov., Campylobacter porcelli sp. nov., and Campylobacter vicugnae sp. nov., three novel Campylobacter lanienae-like species recovered from swine, small ruminants, and camelids.

In a previous study characterizing Campylobacter strains deficient in selenium metabolism, 50 strains were found to be similar to, but distinct from, the selenonegative species Campylobacter lanienae. Initial characterization based on multilocus sequence typing and the phylogeny of a set of 20 core genes determined that these strains form three putative taxa within the selenonegative cluster. A polyphasic study was undertaken here to further clarify their taxonomic position within the genus. The 50 selenonegative strains underwent phylogenetic analyses based on the sequences of the 16S rRNA gene and an expanded set of 330 core genes. Standard phenotypic testing was also performed. All strains were microaerobic and anaerobic, Gram-negative, spiral or curved cells with some displaying coccoid morphologies. Strains were motile, oxidase, catalase, and alkaline phosphatase positive, urease negative, and reduced nitrate. Strains within each clade had unique phenotypic profiles that distinguished them from other members of the genus. Core genome phylogeny clearly placed the 50 strains into three clades. Pairwise average nucleotide identity and digital DNA-DNA hybridization values were all below the recommended cut-offs for species delineation with respect to C. lanienae and other related Campylobacter species. The data presented here clearly show that these strains represent three novel species within the genus, for which the names Campylobacter devanensis sp. nov. (type strain RM3662T=LMG 33097T=NCTC 15074T), Campylobacter porcelli sp. nov. (type strain RM6137T=LMG 33098T=CCUG 77054T=NCTC 15075T) and Campylobacter vicugnae sp. nov. (type strain RM12175T=LMG 33099T=CCUG 77055T=NCTC 15076T) are proposed.

Erratum: Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

This corrects the article 10.3791/66058.

Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.

Oxford Nanopore Technologies (ONT) sequencing is a promising technology. We assessed the performance of the new ONT R10 flowcells and V14 rapid sequencing chemistry for Mtb whole genome sequencing of Mycobacterium tuberculosis (Mtb) DNA extracted from clinical primary liquid cultures (CPLCs). Using the recommended protocols for MinION Mk1C, R10.4.1 MinION flowcells, and the ONT Rapid Sequencing Kit V14 on six CPLC samples, we obtained a pooled library yield of 10.9 ng/µl, generated 1.94 Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all generated reads met the Phred Quality score threshold (>8). To assess if the low data output and sequence quality were due to impurities present in DNA extracted directly from CPLCs, we added a pre-library preparation bead-clean-up step and included purified DNA obtained from an Mtb subculture as a control sample in a second sequencing run. The library yield for DNA extracted from four CPLCs and one Mtb subculture (control) was similar (10.0 ng/µl), 2.38 Gb of bases and 822k reads were produced. The quality was slightly better with 66% of the produced reads having a Phred Quality >8. A third run of DNA from six CPLCs with bead clean-up pre-processing produced a low library yield (±1 Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10× was only achieved for five of 17 (29%) sequenced libraries. Compared to Illumina WGS of the same samples, accurate lineage predictions and full drug resistance profiles from the generated ONT data could not be determined by TBProfiler. Further optimization of the V14 ONT rapid sequencing chemistry and library preparation protocol is needed for clinical Mtb WGS applications.

Lacticaseibacillus salsurivasis sp. nov. and Companilactobacillus muriivasis sp. nov., Isolated from Traditional Chinese Pickle.

Three Gram-stain-positive bacterial strains were isolated from traditional Chinese pickle and characterized using a polyphasic taxonomic approach. Results of 16S rRNA gene sequence analysis indicated that strain 74-4T was most closely related to the type strains of Lacticaseibacillus suibinensis and Lacticaseibacillus suilingensis, having 99.9% and 100% 16S rRNA gene sequence similarities, respectively, and that strains 419-1.2T and 262-4 were most closely related to the type strains of Companilactobacillus heilongjiangensis, Companilactobacillus nantensis, Companilactobacillus huachuanensis, and Companilactobacillus nuruki, having 98.5-99.7% 16S rRNA gene sequence similarities. The phylogenomic trees indicated that strain 74-4T was related to the type strains of L. suibinensis and L. suilingensis, and that strains 419-1.2T and 262-4 were related to the type strains of C. heilongjiangensis, C. nantensis, C. huachuanensis, and Companilactobacillus zhachilii. The ANI and dDDH values between strain 74-4T and type strains of phylogenetically related species were less than 92.7% and 49.9%, respectively. The ANI and dDDH values between strains 419-1.2T and 262-4 and type strains of phylogenetically related species were less than 93.4% and 51.7%, respectively. Based upon the data of polyphasic characterization obtained in the present study, two novel species, Lacticaseibacillus salsurivasis sp. nov. and Companilactobacillus muriivasis sp. nov., are proposed and the type strains are 74-4T (= JCM 35890T = CCTCC AB 2022414T) and 419-1.2T (= JCM 35891T = CCTCC AB 2022413T), respectively.

Extraction of genomic DNA for sequencing from snail Helix lucorum.

In the past, there were no easily distinct and recognizable features as a guide for precise clinical and genetic diagnosis of cases with chromosome microdeletions involving 15q26 including CHD2,. The present study analysed the clinical data and collected venous blood samples from a pediatric patient and his healthy family members for DNA testing. The whole-exome sequencing was performed by the next-generation sequencing (NGS). Chromosomal copy-number variations were tested based on NGS. We present a review of all cases with chromosome microdeletions affecting CHD2. A novel de novo 5.82-Mb deletion at 15q25.3-15q26.1 including CHD2 was identified in our patient who is an 11.6-year-old boy. We first found surprising efficacy of lamotrigine in controlling intractable drop seizures in the individual. These cases have development delay, behavioural problems, epilepsy, variable multiple anomalies, etc. Phenotypes of individuals with deletions involving 15q26 including CHD2 are highly variable with regard to facial features and multiple developmental anomalies. We first found the special clinical entity of development delay, behavioural problems, epilepsy, variable skeletal and muscular anomalies, abnormalities of variable multiple systems and characteristic craniofacial phenotypes in patients with chromosome microdeletions involving CHD2. The larger deletions involving 15q26 including CHD2 tend to cause the classical phenotype. A distinctive craniofacial appearance of the classical phenotype is midface hypoplasia and perifacial protrusion.

Klenkia sesuvii sp. nov., isolated from leaves of halophyte Sesuvium portulacastrum.

During a study on the diversity of culturable actinobacteria from coastal halophytes in Thailand, strain LSe6-5T was isolated from leaves of sea purslane (Sesuvium portulacastrum L.), and a polyphasic approach was employed to determine its taxonomic position. The 16S rRNA gene sequences analysis indicated that the strain was most closely related to Klenkia brasiliensis Tu 6233T (99.2 %), Klenkia marina YIM M13156T (99.1 %), and Klenkia terrae PB261T (98.7 %). The genome of strain LSe6-5T was estimated to be 4.33 Mbp in size, with DNA G+C contents of 74.3%. A phylogenomic tree based on whole-genome sequences revealed that strain LSe6-5T formed a clade with Klenkia marina DSM 45722T, indicating their close relationship. However, the average nucleotide identity (ANI)-blast, ANI-MUMmer, and dDDH values between strain LSe6-5T with K. marina DSM 45722T (87.1, 88.9, and 33.0 %) were below the thresholds of 95-96 % ANI and 70 % dDDH for identifying a novel species. Furthermore, strain LSe6-5T showed morphological and chemotaxonomic characteristics of the genus Klenkia. Cells were motile, rod-shaped, and Gram-stain-positive. Optimal growth of strain LSe6-5T occurred at 28 °C, pH 7.0, and 0-3 % NaCl. The whole-cell hydrolysates contained meso-diaminopimelic acid as the diagnostic diamino acid, with galactose, glucose, mannose, and ribose as whole-cell sugars. The predominant menaquinones were MK-9(H4) and MK-9(H0). The polar lipid profile was composed of diphosphatidylglycerol, hydroxyphosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, an unidentified phospholipid, and an unidentified lipid. Major cellular fatty acids were iso-C15 : 0, iso-C16 : 0, and iso-C17 : 0. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, it is supported that strain LSe6-5T represents a novel species of the genus Klenkia, for which the name Klenkia sesuvii sp. nov. is proposed. The type strain is strain LSe6-5T (=TBRC 16417T= NBRC 115929T).

Roseinatronobacter alkalisoli sp. nov., an alkaliphilic bacterium isolated from soda soil, and genome-based reclassification of the genera Rhodobaca and Roseinatronobacter.

The genera Rhodobaca and Roseinatronobacter are phylogenetically related genera within the family Paracoccaceae. Species of these genera were described using 16S rRNA gene-based phylogeny and phenotypic characteristics. However, the 16S rRNA gene identity and phylogeny reveal the controversy of the taxonomic status of these two genera. In this work, we examined the taxonomic positions of members of both genera using 16S rRNA gene phylogeny, phylogenomic analysis and further validated using overall genome-related indexes, including digital DNA-DNA hybridization, average nucleotide identity, average amino acid identity and percentage of conserved proteins. Based on phylogenetic and phylogenomic results, the current four species of the two genera clustered tightly into one clade with high bootstrap values, suggesting that the genus Rhodobaca should be merged with Roseinatronobacter. In addition, a novel species isolated from a soda soil sample collected from Anda City, PR China, and designated as HJB301T was also described. Phenotypic, chemotaxonomic, genomic and phylogenetic properties suggested that strain HJB301T (=CCTCC AB 2021113T=KCTC 82977T) represents a novel species of the genus Roseinatronobacter, for which the name Roseinatronobacter alkalisoli sp. nov. is proposed.

Paraburkholderia largidicola sp. nov., a gut symbiont of the bordered plant bug Physopelta gutta.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated strains F2T and PGU16, were isolated from the midgut crypts of the bordered plant bug Physopelta gutta, collected in Okinawa prefecture, Japan. Although these strains were derived from different host individuals collected at different times, their 16S rRNA gene sequences were identical and showed the highest similarity to Paraburkholderia caribensis MWAP64T (99.3 %). The genome of strain F2T consisted of two chromosomes and two plasmids, and its size and G+C content were 9.28 Mb and 62.4 mol% respectively; on the other hand, that of strain PGU16 consisted of two chromosomes and three plasmids, and its size and G+C content were 9.47 Mb and 62.4 mol%, respectively. Phylogenetic analyses revealed that these two strains are members of the genus Paraburkholderia. The digital DNA-DNA hybridization value between these two strains was 92.4 %; on the other hand, the values between strain F2T and P. caribensis MWAP64T or phylogenetically closely related Paraburkholderia species were 44.3 % or below 49.1 %. The predominant fatty acids of both strains were C16 : 0, C17 : 0 cyclo, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), and C19 : 0 cyclo ω8c, and their respiratory quinone was ubiquinone 8. Based on the above genotypic and phenotypic characteristics, strains F2T and PGU16 represent a novel species of the genus Paraburkholderia for which the name Paraburkholderia largidicola sp. nov. is proposed. The type strain is F2T (=NBRC 115765T=LMG 32765T).

Roseateles caseinilyticus sp. nov. and Roseateles cellulosilyticus sp. nov., isolated from rice paddy field soil.

Two novel Gram-stain-negative strains designated P7T and P8T, were isolated from the soil of a paddy field in Goyang, Republic of Korea, and identified as new species within the genus Roseateles through a polyphasic taxonomic approach. These aerobic, rod-shaped, non-sporulating strains demonstrated optimal growth at 30 °C, pH 7, and in the absence of NaCl (0% w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated close relationships with Roseateles saccharophilus DSM654T (98.7%) and Roseateles puraquae CCUG 52769T (98.96%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolates with the most closely related strains with publicly available whole genomes were 82.0-85.5% and 25.0-30.2%, respectively. The predominant fatty acids identified were C16:0 and summed feature 3 (composed of C16:1 ω6c and/or C16:1 ω7c), with minor amounts of C12:0, C10:0 3-OH and summed feature 8 (composed of C18:1 ω7c and/or C18:1 ω6c; 26.4%). Ubiquinone 8 was the main quinone, and the polar lipid profile included phosphatidylethanolamine, phosphatidylglycerol, two unidentified phosphoaminolipids, one unidentified phosphoglycolipid, three unidentified phospholipids, and one unidentified aminolipid. The draft genome sequences revealed genomic DNA G + C contents of 70.1% for P7T and 68.2% for P8T. Comprehensive physiological, biochemical, and 16S rRNA sequence analyses confirm these isolates as novel species of the genus Roseateles, proposed to be named Roseateles caseinilyticus sp. nov for strain P7T (= KACC 22504T = TBRC 15694T) and Roseateles cellulosilyticus sp. nov. for strain P8T (= KACC 22505T = TBRC 15695T).