Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

Establishing reference sequences for each clade of SARS-CoV-2 to provide a basis for virus variation and function research.

Coronavirus disease 2019 (COVID-19) is a severe respiratory disease caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As the COVID-19 pandemic continues, mutations of SARS-CoV-2 accumulate. These mutations may not only make the virus spread faster, but also render current vaccines less effective. In this study, we established a reference sequence for each clade defined using the GISAID typing method. Homology analysis of each reference sequence confirmed a low mutation rate for SARS-CoV-2, with the latest clade GRY having the lowest homology with other clades (99.89%-99.93%), and the homology between other clade being greater than or equal to 99.95%. Variation analyses showed that the earliest genotypes S, V, and G had 2, 3, and 3 characterizing mutations in the genome respectively. The G-derived clades GR, GH, and GV had 5, 6, and 13 characterizing mutations in the genome respectively. A total of 28 characterizing mutations existed in the genome of the latest clades GRY. In addition, we found differences in the geographic distribution of different clades. G, GH, and GR are popular in the USA, while GV and GRY are common in the UK. Our work may facilitate the custom design of antiviral strategies depending on the molecular characteristics of SARS-CoV-2.

Publicly Available and Validated DNA Reference Sequences Are Critical to Fungal Identification and Global Plant Protection Efforts: A Use-Case in Colletotrichum.

Publicly available and validated DNA reference sequences useful for phylogeny estimation and identification of fungal pathogens are an increasingly important resource in the efforts of plant protection organizations to facilitate safe international trade of agricultural commodities. Colletotrichum species are among the most frequently encountered and regulated plant pathogens at U.S. ports-of-entry. The RefSeq Targeted Loci (RTL) project at NCBI (BioProject no. PRJNA177353) contains a database of curated fungal internal transcribed spacer (ITS) sequences that interact extensively with NCBI Taxonomy, resulting in verified name-strain-sequence type associations for >12,000 species. We present a publicly available dataset of verified and curated name-type strain-sequence associations for all available Colletotrichum species. This includes an updated GenBank Taxonomy for 238 species associated with up to 11 protein coding loci and an updated RTL ITS dataset for 226 species. We demonstrate that several marker loci are well suited for phylogenetic inference and identification. We improve understanding of phylogenetic relationships among verified species, verify or improve phylogenetic circumscriptions of 14 species complexes, and reveal that determining relationships among these major clades will require additional data. We present detailed comparisons between phylogenetic and similarity-based approaches to species identification, revealing complex patterns among single marker loci that often lead to misidentification when based on single-locus similarity approaches. We also demonstrate that species-level identification is elusive for a subset of samples regardless of analytical approach, which may be explained by novel species diversity in our dataset and incomplete lineage sorting and lack of accumulated synapomorphies at these loci.

Breed-specific reference sequence optimized mapping accuracy of NGS analyses for pigs.

BACKGROUND: Reference sequences play a vital role in next-generation sequencing (NGS), impacting mapping quality during genome analyses. However, reference genomes usually do not represent the full range of genetic diversity of a species as a result of geographical divergence and independent demographic events of different populations. For the mitochondrial genome (mitogenome), which occurs in high copy numbers in cells and is strictly maternally inherited, an optimal reference sequence has the potential to make mitogenome alignment both more accurate and more efficient. In this study, we used three different types of reference sequences for mitogenome mapping, i.e., the commonly used reference sequence (CU-ref), the breed-specific reference sequence (BS-ref) and the sample-specific reference sequence (SS-ref), respectively, and compared the accuracy of mitogenome alignment and SNP calling among them, for the purpose of proposing the optimal reference sequence for mitochondrial DNA (mtDNA) analyses of specific populations RESULTS: Four pigs, representing three different breeds, were high-throughput sequenced, subsequently mapping reads to the reference sequences mentioned above, resulting in a largest mapping ratio and a deepest coverage without increased running time when aligning reads to a BS-ref. Next, single nucleotide polymorphism (SNP) calling was carried out by 18 detection strategies with the three tools SAMtools, VarScan and GATK with different parameters, using the bam results mapping to BS-ref. The results showed that all eighteen strategies achieved the same high specificity and sensitivity, which suggested a high accuracy of mitogenome alignment by the BS-ref because of a low requirement for SNP calling tools and parameter choices. CONCLUSIONS: This study showed that different reference sequences representing different genetic relationships to sample reads influenced mitogenome alignment, with the breed-specific reference sequences being optimal for mitogenome analyses, which provides a refined processing perspective for NGS data.

Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut.

BACKGROUND: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge. RESULTS: A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes. CONCLUSIONS: The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

The establishment of reference sequence for SARS-CoV-2 and variation analysis.

Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.

A new hepatitis B virus e antigen-negative strain gene used as a reference sequence in an animal model.

Infection with hepatitis B virus (HBV) e-antigen (HBeAg)-negative strains is increasingly prevalent. Currently, detailed information of the obtained natural HBV strain is not available except for the B genotype and HBeAg-negative. The aim of the present study was to characterize the natural genetic variation of the HBeAg-negative strain and investigate its function. The genic sequence was determined using Sanger sequencing, and compared to related sequences using alignment and phylogenetic analysis. In vivo, virus-specific serum markers were investigated in CBA/CaJ mice. The sequence had a full genome length of 3215 nucleotides. Sites 122, 125, 127, and 160 in S regions were identified as lysine, threonine, proline, and lysine respectively. The main four point variants including A1762T, G1764A, G1896A, and G1899A were detected in the full-length genome. The genotype of the sequence was B, with sub-genotype B2 and serological subtype adw2. The characterize of the natural genetic variation strain showed no reported drug-resistant variant in P region and no reported immune escape site in S region. The strain will increase viral replication and infection for mutations A1762T and G1764A in the basal core promoter region, and mutations G1896A and G1899A in the pre-core region. The G1896A variant resulted in a premature stop codon and abolished HBeAg expression. HBsAg persisted for 26 weeks and HBeAg was still negative in CBA/CaJ mice. The present sequence is representative of the HBeAg-negative genome and may serve as a valuable reference for studying HBeAg-negative strains. The present findings were successfully verified in CBA/CaJ mice, demonstrating good applicability of the sequence.

The Bermuda Triangle: The Pragmatics, Policies, and Principles for Data Sharing in the History of the Human Genome Project.

The Bermuda Principles for DNA sequence data sharing are an enduring legacy of the Human Genome Project (HGP). They were adopted by the HGP at a strategy meeting in Bermuda in February of 1996 and implemented in formal policies by early 1998, mandating daily release of HGP-funded DNA sequences into the public domain. The idea of daily sharing, we argue, emanated directly from strategies for large, goal-directed molecular biology projects first tested within the "community" of C. elegans researchers, and were introduced and defended for the HGP by the nematode biologists John Sulston and Robert Waterston. In the C. elegans community, and subsequently in the HGP, daily sharing served the pragmatic goals of quality control and project coordination. Yet in the HGP human genome, we also argue, the Bermuda Principles addressed concerns about gene patents impeding scientific advancement, and were aspirational and flexible in implementation and justification. They endured as an archetype for how rapid data sharing could be realized and rationalized, and permitted adaptation to the needs of various scientific communities. Yet in addition to the support of Sulston and Waterston, their adoption also depended on the clout of administrators at the US National Institutes of Health (NIH) and the UK nonprofit charity the Wellcome Trust, which together funded 90% of the HGP human sequencing effort. The other nations wishing to remain in the HGP consortium had to accommodate to the Bermuda Principles, requiring exceptions from incompatible existing or pending data access policies for publicly funded research in Germany, Japan, and France. We begin this story in 1963, with the biologist Sydney Brenner's proposal for a nematode research program at the Laboratory of Molecular Biology (LMB) at the University of Cambridge. We continue through 2003, with the completion of the HGP human reference genome, and conclude with observations about policy and the historiography of molecular biology.

LSDBs and How They Have Evolved.

Locus specific databases (LSDBs) make a key contribution to our understanding of heritable and acquired human disorders, disease susceptibility, and adverse drug reactions. As data have accumulated in LSDBs, a greater reliance on their use has arisen in clinical practice. Even though LSDBs have existed in recognizable form for only a quarter of a century, their origin lies in the manual cataloging of data that began around 50 years ago. Analysis and recording of sequence variation in the globin genes, and the proteins which they encode, can confidently be said to be the foundation for what we now refer to as LSDBs. Their growth over the years has primarily been underpinned by software developments and the advent of the World Wide Web. However, it is also important to recognize the evolution of reporting standards and reference sequences, without which accurate and consistent reporting of sequence variants would be impossible. Nowadays, LSDBs exist for many human protein-coding genes and the focus of efforts has moved toward minor tidying up of the variant reporting nomenclature and processes for assuring the completeness, correctness, and consistency of the data. The next 25 years will doubtless witness further developments in the evolution of LSDBs.

Comparative BAC-based physical mapping of Oryza sativa ssp. indica var. 93-11 and evaluation of the two rice reference sequence assemblies.

Reference sequences are sequences that are used for public consultation, and therefore must be of high quality. Using the whole-genome shotgun/next-generation sequencing approach, many genome sequences of complex higher plants have been generated in recent years, and are generally considered reference sequences. However, none of these sequences has been experimentally evaluated at the whole-genome sequence assembly level. Rice has a relatively simple plant genome, and the genome sequences for its two sub-species obtained using different sequencing approaches were published approximately 10 years ago. This provides a unique system for a case study to evaluate the qualities and utilities of published plant genome sequences. We constructed a robust BAC physical map embedding a large number of BAC end sequences forrice variety 93-11. Through BAC end sequence alignments and tri-assembly comparisons of the 93-11 physical map and the two reference sequences, we found that the Nipponbare reference sequence generated using the clone-by-clone approach has a high quality but still contains small artifact inversions and missing sequences. In contrast, the 93-11 reference sequence generated using the whole-genome shotgun approach contains many large and varied assembly errors, such as inversions, duplications and translocations, as well as missing sequences. The 93-11 physical map provides an invaluable resource for evaluation and improvements toward completion of both Nipponbare and 93-11 reference sequences.

Genomic medicine and risk prediction across the disease spectrum.

Genomic medicine is based on the knowledge that virtually every medical condition, disease susceptibility or response to treatment is caused, regulated or influenced by genes. Genetic testing may therefore add value across the disease spectrum, ranging from single-gene disorders with a Mendelian inheritance pattern to complex multi-factorial diseases. The critical factors for genomic risk prediction are to determine: (1) where the genomic footprint of a particular susceptibility or dysfunction resides within this continuum, and (2) to what extent the genetic determinants are modified by environmental exposures. Regarding the small subset of highly penetrant monogenic disorders, a positive family history and early disease onset are mostly sufficient to determine the appropriateness of genetic testing in the index case and to inform pre-symptomatic diagnosis in at-risk family members. In more prevalent polygenic non-communicable diseases (NCDs), the use of appropriate eligibility criteria is required to ensure a balance between benefit and risk. An additional screening step may therefore be necessary to identify individuals most likely to benefit from genetic testing. This need provided the stimulus for the development of a pathology-supported genetic testing (PSGT) service as a new model for the translational implementation of genomic medicine in clinical practice. PSGT is linked to the establishment of a research database proven to be an invaluable resource for the validation of novel and previously described gene-disease associations replicated in the South African population for a broad range of NCDs associated with increased cardio-metabolic risk. The clinical importance of inquiry concerning family history in determining eligibility for personalized genotyping was supported beyond its current limited role in diagnosing or screening for monogenic subtypes of NCDs. With the recent introduction of advanced microarray-based breast cancer subtyping, genetic testing has extended beyond the genome of the host to also include tumor gene expression profiling for chemotherapy selection. The decreasing cost of next generation sequencing over recent years, together with improvement of both laboratory and computational protocols, enables the mapping of rare genetic disorders and discovery of shared genetic risk factors as novel therapeutic targets across diagnostic boundaries. This article reviews the challenges, successes, increasing inter-disciplinary integration and evolving strategies for extending PSGT towards exome and whole genome sequencing (WGS) within a dynamic framework. Specific points of overlap are highlighted between the application of PSGT and exome or WGS, as the next logical step in genetically uncharacterized patients for whom a particular disease pattern and/or therapeutic failure are not adequately accounted for during the PSGT pre-screen. Discrepancies between different next generation sequencing platforms and low concordance among variant-calling pipelines caution against offering exome or WGS as a stand-alone diagnostic approach. The public reference human genome sequence (hg19) contains minor alleles at more than 1 million loci and variant calling using an advanced major allele reference genome sequence is crucial to ensure data integrity. Understanding that genomic risk prediction is not deterministic but rather probabilistic provides the opportunity for disease prevention and targeted treatment in a way that is unique to each individual patient.

A Pseudo-nitzschia metabarcoding approach with a calibrated ITS1 reference sequence database applied in the Taiwan Strait.

Pseudo-nitzschia is a cosmopolitan phytoplankton genus of which some species can form blooms and produce the neurotoxin domoic acid (DA). Identification of Pseudo-nitzschia is generally based on field material or strains followed by morphological and/or molecular characterization. However, this process is time-consuming and laborious, and can not obtain a relatively complete and reliable profile of the Pseudo-nitzschia community, because species with low abundance in the field or potentially unavailable for culturing may easily be overlooked. In the present study, specific ITS primer sets were designed and evaluated using in silico matching. The primer set ITS-84F/456R involving the complete ITS1 region was found optimal. Based on matching with a Pseudo-nitzschia ITS1 reference sequence database carefully-calibrated in this study, a metabarcoding approach using annotated amplicon sequence variants (ASV) was applied in the Taiwan Strait of the East China Sea during two cruises in the spring and summer of 2019. In total, 48 Pseudo-nitzschia species/phylotypes including 36 known and 12 novel were uncovered, and verified by haplotype networks, ITS2 secondary structure comparisons and divergence analyses. Correlation analyses revealed that temperature was a key factor affecting the seasonal variation of the Pseudo-nitzschia community. This study provides an overview of the Pseudo-nitzschia community in the Taiwan Strait, with new insights into the diversity. The developed metabarcoding approach may be used elsewhere as a standard reference for accurate annotation of Pseudo-nitzschia.

Creating, curating and evaluating a mitogenomic reference database to improve regional species identification using environmental DNA.

Species detection using eDNA is revolutionizing global capacity to monitor biodiversity. However, the lack of regional, vouchered, genomic sequence information-especially sequence information that includes intraspecific variation-creates a bottleneck for management agencies wanting to harness the complete power of eDNA to monitor taxa and implement eDNA analyses. eDNA studies depend upon regional databases of mitogenomic sequence information to evaluate the effectiveness of such data to detect and identify taxa. We created the Oregon Biodiversity Genome Project to create a database of complete, nearly error-free mitogenomic sequences for all of Oregon's fishes. We have successfully assembled the complete mitogenomes of 313 specimens of freshwater, anadromous and estuarine fishes representing 24 families, 55 genera and 129 species and lineages. Comparative analyses of these sequences illustrate that many regions of the mitogenome are taxonomically informative, that the short (~150 bp) mitochondrial 'barcode' regions typically used for eDNA assays do not consistently diagnose for species and that complete single or multiple genes of the mitogenome are preferable for identifying Oregon's fishes. This project provides a blueprint for other researchers to follow as they build regional databases, illustrates the taxonomic value and limits of complete mitogenomic sequences and offers clues as to how current eDNA assays and environmental genomics methods of the future can best leverage this information.

Exploring the key genomic variation in monkeypox virus during the 2022 outbreak.

BACKGROUND: In 2022, a global outbreak of monkeypox occurred with a significant shift in its epidemiological characteristics. The monkeypox virus (MPXV) belongs to the B.1 lineage, and its genomic variations that were linked to the outbreak were investigated in this study. Previous studies have suggested that viral genomic variation plays a crucial role in the pathogenicity and transmissibility of viruses. Therefore, understanding the genomic variation of MPXV is crucial for controlling future outbreaks. METHODS: This study employed bioinformatics and phylogenetic approaches to evaluate the key genomic variation in the B.1 lineage of MPXV. A total of 979 MPXV strains were screened, and 212 representative strains were analyzed to identify specific substitutions in the viral genome. Reference sequences were constructed for each of the 10 lineages based on the most common nucleotide at each site. A total of 49 substitutions were identified, with 23 non-synonymous substitutions. Class I variants, which had significant effects on protein conformation likely to affect viral characteristics, were classified among the non-synonymous substitutions. RESULTS: The phylogenetic analysis revealed 10 relatively monophyletic branches. The study identified 49 substitutions specific to the B.1 lineage, with 23 non-synonymous substitutions that were classified into Class I, II, and III variants. The Class I variants were likely responsible for the observed changes in the characteristics of circulating MPXV in 2022. These key mutations, particularly Class I variants, played a crucial role in the pathogenicity and transmissibility of MPXV. CONCLUSION: This study provides an understanding of the genomic variation of MPXV in the B.1 lineage linked to the recent outbreak of monkeypox. The identification of key mutations, particularly Class I variants, sheds light on the molecular mechanisms underlying the observed changes in the characteristics of circulating MPXV. Further studies can focus on functional domains affected by these mutations, enabling the development of effective control strategies against future monkeypox outbreaks.

Standard reference sequences for submission of HLA genotyping for the 18th International HLA and Immunogenetics Workshop.

The International human leukocyte antigen (HLA) and Immunogenetics Workshops (IHIWs) have fostered international collaborations of researchers and experts in the fields of HLA, histocompatibility and immunology. These IHIW collaborations have comprised many projects focused on achieving a variety of specific goals. The international and collaborative nature of these projects necessitates the collection and analysis of complex data generated in multiple laboratories, often using multiple methods of acquisition. Collection and storage of these data in a consistent way adds value to IHIW projects, which can be extended to future work. DNA-based genotyping data, especially HLA genotyping data, can be transmitted in the form of a Histoimmunogenetics Markup Language (HML) document. HML facilitates clear communication of a genotype and supporting metadata, such as, sequencing platform, laboratory assays, consensus sequence, and interpretation. Sequence information can be reported relative to known reference sequences, which add meaning and context to genotypes. Selecting the correct reference sequence for a given allele sequence is nuanced, and guidelines have emerged through collaborative community efforts such as Data Standards Hackathons. Here, we describe the guidelines established for the selection of reference sequences to be used in transmission of HLA (and MICA/MICB) genotyping data for the 18th IHIW.

Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety.

Potato (Solanum tuberosum L.) is one of the most important crops with a worldwide production of 370 million metric tons. The objectives of this study were (1) to create a high-quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (2) to evaluate the new assembly's usefulness for various genomic methods; and (3) to assess the performance of phasing in diploid and tetraploid clones, using linked-read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg1. Comparisons of the new dAg1_v1.0 sequence to other potato genome sequences point out the high divergence between the different potato varieties and illustrate the potential of using dAg1_v1.0 sequence in breeding applications.

Non-Redundant tRNA Reference Sequences for Deep Sequencing Analysis of tRNA Abundance and Epitranscriptomic RNA Modifications.

Analysis of RNA by deep-sequencing approaches has found widespread application in modern biology. In addition to measurements of RNA abundance under various physiological conditions, such techniques are now widely used for mapping and quantification of RNA modifications. Transfer RNA (tRNA) molecules are among the frequent targets of such investigation, since they contain multiple modified residues. However, the major challenge in tRNA examination is related to a large number of duplicated and point-mutated genes encoding those RNA molecules. Moreover, the existence of multiple isoacceptors/isodecoders complicates both the analysis and read mapping. Existing databases for tRNA sequencing provide near exhaustive listings of tRNA genes, but the use of such highly redundant reference sequences in RNA-seq analyses leads to a large number of ambiguously mapped sequencing reads. Here we describe a relatively simple computational strategy for semi-automatic collapsing of highly redundant tRNA datasets into a non-redundant collection of reference tRNA sequences. The relevance of the approach was validated by analysis of experimentally obtained tRNA-sequencing datasets for different prokaryotic and eukaryotic model organisms. The data demonstrate that non-redundant tRNA reference sequences allow improving unambiguous mapping of deep sequencing data.

Reconstructing protein-coding sequences from ancient DNA.

Obtaining information about functional details of proteins of extinct species is of critical importance for a better understanding of the real-life appearance, behavior and ecology of these lost entries in the book of life. In this chapter, we discuss the possibilities to retrieve the necessary DNA sequence information from paleogenomic data obtained from fossil specimens, which can then be used to express and subsequently analyze the protein of interest. We discuss the problems specific to ancient DNA, including miscoding lesions, short read length and incomplete paleogenome assemblies. Finally, we discuss an alternative, but currently rarely used approach, direct PCR amplification, which is especially useful for comparatively short proteins.

Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments.

High-throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species-level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum-specific species-level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read-merging and two clustering algorithms and the cluster-free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus-level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species-level detections, which were, however, associated with higher error rates compared to ASVs. Genus-level assignments using ASVs exhibited higher accuracy and lower error rates compared to species-level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.

There is no Difference Between Sequences of HIV-1 Infected Patients with Stable Clinical Status and HIV-1 Reference Sequence.

BACKGROUND: The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the past few years. HIV-1 genome sequences are pivotal for large-scale studies of inter- and intra-host evolution. To understand the molecular difference between reference HIV-1 isolate and two HIV-1 infected patients in Iran, we conducted this study to analyze some genome segments of Iranian HIV-1 isolates. METHODS: Two HIV-1-infected individuals who were under antiretroviral therapy (ARV) for 8 years with stable clinical status were enrolled. The patient's plasma samples were used for the Gag-Pol genome sequences (4500 nt). The phylogenetic tree and similarity plotty were obtained based on Gag-Pol sequences. RESULTS: Both HIV-1-infected isolates belonged to CRF35_AD subtype even though one of them had drug resistance. The HIV genome and protein sequences showed no clear difference between genome and protein sequences of our samples and the reference sequence. CONCLUSIONS: Our patient's stable clinical status had no connection to genome sequence; which could be owing to immunological factors or other patient's mode which are still unknown.