Chimeric Sequences Research Articles

Free access of published DNA sequences facilitates regular control of (meta‐) data quality – an example from shorebird mitogenomes (Aves, Charadriiformes: Charadrius)

Online repositories of DNA sequences are a rich and indispensable source of comparative data for biodiversity research and taxonomic studies. Despite increasingly high data quality of published sequences and associated metadata, particular attention should be paid to taxonomic assignment of DNA sequences, in particular if voucher specimens are not available or cannot be examined. In this study, two nearly identical mitogenomes of two distinctive plover species (Charadrius alexandrinus and Charadrius placidus) were re‐analysed and compared with a comprehensive dataset of DNA‐barcode sequences (cytochrome‐oxidase subunit 1, COI) for 55 shorebird species. Phylogenetic analysis separated the two plover species into two reciprocally monophyletic clades that differed by mean p‐distances of 11.5–14.7%; however, the COI sequence from the C. placidus mitogenome was nested in the Kentish Plover clade (C. alexandrinus). A similar mismatch was found for another DNA‐barcode sequence from a Charadrius mongolus mitogenome that clustered with one of two clades of Charadrius leschenaultii in the COI tree. These results strongly suggest that, to date, two of seven mitogenomes published for Charadriidae are not representative of the taxon names to which the respective GenBank entries were assigned. Only a few DNA‐barcode sequences were associated with outdated taxonomy, while others were suspected to be chimeric sequences. Thus, free access to digital sequence information is a key factor for steady improvement of data quality in online repositories via swarm intelligence of the scientific community.

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks.

Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

Intronic LINE-1 insertion in SLCO1B3 as a highly prevalent cause of rotor syndrome in East Asian population.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, thatwas generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

Revealing protein-protein interactions at the transcriptome scale by sequencing

We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated invivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.

Abstract 241: An efficient probe design algorithm for direct fusion targeting from RNA

Abstract Background: The use of sequencing technologies to detect gene fusions (GFs) from RNA shows promising results for the future of cancer diagnosis and treatment. Major obstacles for this approach include target design and lack of well-curated databases of RNA breakpoints. Currently, off-the-shelf designs include full transcript targeting that results in massive and costly amounts of data. Directly targeting the known GFs from RNA by designing probes targeting the fusion junction sequence is studied here as an alternative to whole-exome sequencing (WES). We present notably a novel algorithm capable of designing the probes to accurately target the desired fusions from RNA. Methods: For a given GF detected either from DNA or from RNA, the algorithm is as follows: (1) Collect gene and isoform information for both partners from seven public databases; (2) For each candidate pair of isoforms, locate where the breakpoints will be observed and assign a score based on various criteria such as sequence completion, coding information, transcript support level, % identity with and % visible on hg38; (3) Select the top scoring pair of transcripts and extract the chimeric probe sequence. Two sets of probes extracted with this protocol targeting 524 and 1632 known GFs were synthetized and tested on several samples (Table 1). The Agilent SureSelect Human All Exon V6 capture kit was used to compare targeting efficiency against WES. Results: Targeted enrichment of a SeraSeq control showed a 5 to 20 fold increase in supporting evidence over WES. On 10 clinical samples, we observed 10-30x increase in supporting reads. A higher sensitivity is observed in both cases. Conclusion: We developed a novel algorithm capable of accurately identifying the most likely location of an RNA fusion junction and generating the probe sequences for oligo synthesis. This method not only enriches for more supporting data but also reduces the associated costs. Average number of supporting reads per fusion per million reads for WES & direct targetingSampleKnown FusionWES (A)WES (B)524 TF (A)524 TF (B)1632 TF (A)1632 TF (B)SeraSeq 0710-0496CCDC6→RET8102703558178CD74→ROS13581592832246250EGFR→SEPTIN1418172343159180FGFR3→BAIAP2L114542832612572FGFR3→TACC3239861879270203LMNA→NTRK123112152807167PAX8→PPARG29191932466662SLC34A2→ROS1102217642589142SLC45A3→BRAF1115433420141105TFG→NTRK13540275377128132TMPRSS2→ERG0055934817079TPM3→NTRK11523246359106117Avg. SeraSeq12 Fusions1821373430132116Clinical S1EML4→ALK2515344NA81NAClinical S2EWSR1→FLI15738514NA111NAClinical S3TES→MET2030115NA75NAClinical S4EZR→ROS14311,059NA266NAClinical S5SDC4→ROS1553,354NA1,082NAClinical S6SH3BP5→PPARG004NA1NAClinical S7H2BC21→NTRK11118NA4NAClinical S8COL1A1→PDGFB2092505,530NA2,289NAClinical S9KIF5B→RET2424437NA174NAClinical S10POC1B→GLI1158416NA161NAAvg. Clinical10 Fusions36401,179NA424NA Citation Format: Christophe N. Magnan, Steven P. Rivera, Fernando J. Lopez-Diaz, Chen-Yin Ou, Kenneth B. Thomas, Hyunjun Nam, Lawrence M. Weiss, Segun C. Jung, Vincent A. Funari. An efficient probe design algorithm for direct fusion targeting from RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 241.

Phase Resolution of Heterozygous Sites in Diploid Genomes is Important to Phylogenomic Analysis under the Multispecies Coalescent Model.

Genome sequencing projects routinely generate haploid consensus sequences from diploid genomes, which are effectively chimeric sequences with the phase at heterozygous sites resolved at random. The impact of phasing errors on phylogenomic analyses under the multispecies coalescent (MSC) model is largely unknown. Here, we conduct a computer simulation to evaluate the performance of four phase-resolution strategies (the true phase resolution, the diploid analytical integration algorithm which averages over all phase resolutions, computational phase resolution using the program PHASE, and random resolution) on estimation of the species tree and evolutionary parameters in analysis of multilocus genomic data under the MSC model. We found that species tree estimation is robust to phasing errors when species divergences were much older than average coalescent times but may be affected by phasing errors when the species tree is shallow. Estimation of parameters under the MSC model with and without introgression is affected by phasing errors. In particular, random phase resolution causes serious overestimation of population sizes for modern species and biased estimation of cross-species introgression probability. In general, the impact of phasing errors is greater when the mutation rate is higher, the data include more samples per species, and the species tree is shallower with recent divergences. Use of phased sequences inferred by the PHASE program produced small biases in parameter estimates. We analyze two real data sets, one of East Asian brown frogs and another of Rocky Mountains chipmunks, to demonstrate that heterozygote phase-resolution strategies have similar impacts on practical data analyses. We suggest that genome sequencing projects should produce unphased diploid genotype sequences if fully phased data are too challenging to generate, and avoid haploid consensus sequences, which have heterozygous sites phased at random. In case the analytical integration algorithm is computationally unfeasible, computational phasing prior to population genomic analyses is an acceptable alternative. [BPP; introgression; multispecies coalescent; phase; species tree.]

A new type of triploid fish derived from the diploid hybrid crucian carp (♀) × autotetraploid fish (♂)

Based on the allodiploid lineage (WR, 2n ​= ​100, F1–F5) of white crucian carp (Carassius cuvieri, WCC, 2n ​= ​100) (♀) ​× ​red crucian carp (Carassius auratus red var., RCC, 2n ​= ​100) (♂) and the diploid hybrid fish (WR-II, 2n ​= ​100) derived from WR-F1 (♀) ​× ​WCC (♂), in this research, we obtain a new type of triploid fish (WR-III) by the distant hybridization of WR-II (♀) and autotetraploid fish (4nAU, 4n ​= ​200) (♂). We investigate the morphological traits, chromosomal numbers, DNA content, growth rates and 5S rDNA in the WR-III. The results show that the WR-III is triploid fish with 150 chromosomes and they have no barbel and have a gray body color. All the measurable traits of WR-III show hybrid characteristics; and there is a significant difference in the body length/body height between WR-III and 4nAU, which indicating that WR-III has a smaller head and higher body compared with 4nAU; furthermore, the WR-III has a fast growth rate, the average body weights of eight months old WR-III is 487g. The 5s rDNA of the WR-III inherits the 5s rDNA of its parents and has some mutant bases or chimeric sequences, which revealed heredity, variability and heterozygosity in WR-III. The successful production of this triploid fish is of great significance in fish genetic breeding.

Rumen Epithelial Communities Share a Core Bacterial Microbiota: A Meta-Analysis of 16S rRNA Gene Illumina MiSeq Sequencing Datasets.

In this meta-analysis, 17 rumen epithelial 16S rRNA gene Illumina MiSeq amplicon sequencing data sets were analyzed to identify a core rumen epithelial microbiota and core rumen epithelial OTUs shared between the different studies included. Sequences were quality-filtered and screened for chimeric sequences before performing closed-reference 97% OTU clustering, and de novo 97% OTU clustering. Closed-reference OTU clustering identified the core rumen epithelial OTUs, defined as any OTU present in ≥ 80% of the samples, while the de novo data was randomly subsampled to 10,000 reads per sample to generate phylum- and genus-level distributions and beta diversity metrics. 57 core rumen epithelial OTUs were identified including metabolically important taxa such as Ruminococcus, Butyrivibrio, and other Lachnospiraceae, as well as sulfate-reducing bacteria Desulfobulbus and Desulfovibrio. Two Betaproteobacteria OTUs (Neisseriaceae and Burkholderiaceae) were core rumen epithelial OTUs, in contrast to rumen content where previous literature indicates they are rarely found. Two core OTUs were identified as the methanogenic archaea Methanobrevibacter and Methanomethylophilaceae. These core OTUs are consistently present across the many variables between studies which include different host species, geographic region, diet, age, farm management practice, time of year, hypervariable region sequenced, and more. When considering only cattle samples, the number of core rumen epithelial OTUs expands to 147, highlighting the increased similarity within host species despite geographical location and other variables. De novo OTU clustering revealed highly similar rumen epithelial communities, predominated by Firmicutes, Bacteroidetes, and Proteobacteria at the phylum level which comprised 79.7% of subsampled sequences. The 15 most abundant genera represented an average of 54.5% of sequences in each individual study. These abundant taxa broadly overlap with the core rumen epithelial OTUs, with the exception of Prevotellaceae which were abundant, but not identified within the core OTUs. Our results describe the core and abundant bacteria found in the rumen epithelial environment and will serve as a basis to better understand the composition and function of rumen epithelial communities.

Misclassifications in human papillomavirus databases

We assessed the quality of human papillomavirus (HPV) sequences in GenBank by analyzing the possible presence of chimeras, “wrong-assembled” contigs and errors in taxonomy using an open-source script (HPVChimera_Gb) that compared 25 638 HPV-related nucleotide sequences in GenBank with the 221 numbered HPV types and another 220 complete HPV sequences. There were 110 sequences with taxonomy/naming errors (sequences reported as another HPV type than the one they corresponded to) and 1318 possibly chimeric sequences. Manual analysis found plausible explanations for most of them (e.g. sequence covering an integration site) but 114 sequences appeared to be chimeras (96/114 were already flagged as “unverified” by GenBank) and 13 had taxonomy/naming errors. When comparing all correct HPV sequences in GenBank, there appeared to exist about 800 unique putative HPV types. Systematic and regular work towards eliminating chimeric sequences and taxonomy/naming errors could increase the quality and order in HPV research.

Innovative 2'-O-Imino-2-propanoate-Protecting Group for Effective Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for the 2'-hydroxyl function of ribonucleosides is still challenging, particularly when RNA sequences must be of the highest purity for therapeutic applications as nucleic acid-based drugs. A 2'-hydroxyl-protecting group should optimally (i) be easy to install; (ii) allow rapid and efficient incorporation of the 2'-O-protected ribonucleosides into RNA sequences to minimize, to the greatest extent possible, the formation of process-related impurities (e.g., shorter than full-length sequences) during solid-phase synthesis; and (iii) be completely cleaved from RNA sequences without the production of alkylating side products and/or formation of mutagenic nucleobase adducts. The reaction of 2'-O-aminoribonucleosides with ethyl pyruvate results in the formation of stable 2'-O-imino-2-methyl propanoic acid ethyl esters and, subsequently, of the fully protected ribonucleoside phosphoramidite monomers, which are required for the solid-phase synthesis of two chimeric RNA sequences (20-mers) containing the four canonical ribonucleosides. Upon treatment of the RNA sequences with a solution of sodium hydroxide, the 2'-O-imino-2-methyl propanoic acid ethyl ester-protecting groups are saponified to their sodium salts, which after ion exchange underwent quantitative intramolecular decarboxylation under neutral conditions at 65 °C to provide fully deprotected RNA sequences in marginally better yields than those obtained from commercial 2'-O-tert-butyldimethylsilyl ribonucleoside phosphoramidites under highly similar conditions.

Accelerated molecular breeding of novel cytoplasmic male sterility lines in rice using orfH79 or orf290 haplotype

AbstractThe identification and development of new cytoplasmic male sterility (CMS) lines in higher plants are important for the preservation of grain security and the prevention of the homogenisation of hybrid rice. Molecular markers‐assisted selection (MAS) based on CMS‐associated genes or mitochondrial‐specific chimeric sequences are important for rapid and effective breeding of new CMS lines and hybrids. In our study, the distribution and allele variation of orfH79 and orf290 genes were characterised from 273 wild and cultivated rice in the AA genome species. Based on the alignment of amino acid sequences, four accessions with orfH79 and three accessions with orf290 were screened. Four novel CMS lines carrying orfH79 haplotypes and three novel CMS lines carrying orf290 haplotypes were then developed using multiple backcross generations with a maintainer line under MAS. The breeding process used in our study provides an efficient and feasible approach for selecting new CMS lines. CMS lines selected in our study are important for enriching rice germplasm resources and guaranteeing rice breeding programmes.

Recovery of microbial community profile information hidden in chimeric sequence reads

The next frontier in the field of microbiome studies is identification of all microbes present in the microbiome and accurate determination of their abundance such that microbiome profiles can serve as reliable assessments of health or disease status. PCR-based 16S rRNA gene sequencing and metagenome shotgun sequencing technologies are the prevailing approaches used in microbiome analyses. Each poses a number of technical challenges associated with PCR amplification, sample availability, and cost of processing and analysis. In general, results from these two approaches rarely agree completely with each other. Here, we compare these methods utilizing a set of vaginal swab and lavage specimens from a cohort of 42 pregnant women collected for a pilot study exploring the effect of the vaginal microbiome on preterm birth. We generated the microbial community profiles from the sequencing reads of the V3V4 and V4V5 regions of the 16S rRNA gene in the vaginal swab and lavage samples. For a subset of the vaginal samples from 12 subjects, we also performed metagenomic shotgun sequencing analysis and compared the results obtained from the PCR-based sequencing methods. Our findings suggest that sample composition and complexity, particularly at the species level, are major factors that must be considered when analyzing and interpreting microbiome data. Our approach to sequence analysis includes consideration of chimeric reads, by using our chimera-counting BlastBin program, and enables recovery of microbial content information generated during PCR-based sequencing methods, such that the microbial profiles more closely resemble those obtained from metagenomic read-based approaches.

ТАКСОНОМИЧЕСКАЯ И ФУНКЦИОНАЛЬНАЯ ХАРАКТЕРИСТИКА МИКРОБИОТЫ РУБЦА ЛАКТИРУЮЩИХ КОРОВ ПОД ВЛИЯНИЕМ ПРОБИОТИКА ЦЕЛЛОБАКТЕРИНА+

Today rations for dairy cows are designed to provide the highest growth rate and productivity in a short period of time. However, such intensive livestock farming affects, first of all, the health of animals, since metabolic pathways inherent in ruminants are disrupted. The use of 16S metagenomics approaches makes it possible to assess the genetic and metabolic diversity of the bovine microbiome, which allows identifying factors that can contribute to an increase in productivity and an improvement in the health of the host. In the feeding trial, dairy cows were fed with dietary probiotic Cellobacterin+ based on the Enterococcus faecium 1-35 strain (the winter-spring period of 2018, JSC PZ Plamya, Gatchinsky District, Leningrad Province). Two groups of ten Holsteinized black-and-white dairy cows (Bos taurus taurus) of the 2nd and 3rd lactation with an average annual milk yield of 7000-7500 kg were used. The basal diet was 10 kg compound feed, 2 kg yellow corn, 0.5 kg sunflower cake, 0.5 kg rapeseed cake, 1 kg hay, 25 kg grass silage, 1 kg beet molasses, and 0.2 kg MINVIT®-3 (Russia). In the morning, the test cows were fed with dietary Cellobacterin+ (OOO BIOTROF, St. Petersburg) at 40 g per cow. Cicatricial contents (10-50 g) were collected from three cows of each group at the end of the experiment. Fasting blood was taken for biochemical analysis from the tail vein with vacutainers. The blood was analyzed for total protein, total bilirubin, glucose, calcium, phosphorus, urea, reserve alkalinity, ketone bodies. The mass fraction of fat in milk was analyzed according to GOST 5867-90, protein according to GOST 23327-98, and the number of somatic cells according to GOST R 54761-2011. Total DNA from the studied samples was extracted using the Genomic DNA Purification Kit (Fermentas, Inc., Lithuania) according to the attached instructions. Amplification for subsequent NGS sequencing was run (a Veriti Thermal Cycler, Life Technologies, Inc., USA) using the eubacterial primers (IDT) 343F (5´-CTCCTACGGRRSGCAGCAG-3´) and 806R (5´-GGACTANVGGGTWTCTAAT-3´) flanking the V1V3 region of the 16S rRNA gene. Metagenomic sequencing (a MiSeq system, Illumina, Inc., USA) was performed with a MiSeq Reagent Kit v3 (Illumina, Inc., USA). Chimeric sequences were excluded from analysis using the USEARCH 7.0 program (http://drive5.com/usearch/). The processing of the obtained reads using the bioinformatics platform CLC Bio GW 7.0 (Qiagen, the Netherlands) included overlapping, quality filtering (QV > 15), and primer trimming. The taxonomic affiliation of microorganisms to genus was determined using the RDP Classifier program (http://rdp.cme.msu.edu/). Mathematical and statistical processing of the results was carried out using the software packages Microsoft Office Excel 2003, R-Studio (Version 1.1.453) (https://rstudio.com). The mean values (M) and standard errors of the means (±SEM) were calculated. The results were deemed significant at p < 0.05. Analysis of microbial β-diversity of the samples by the principal component method was performed according to the Weighted UniFrac PCoA Emperor method using the QIIME software package. Reconstruction and prediction of the functional content of the metagenome, gene families, and enzymes was performed using the PICRUSt2 software package (v.2.3.0). MetaCyc database (https://metacyc.org/) was used to analyze metabolic pathways and enzymes. Feeding the probiotic had a significant effect (p = 0.049) on an increase in milk yield, as well as on a decrease (p = 0.003) in the somatic cell number in milk by 38,000/ml per cow. The NGS sequencing provided a complete taxonomic and functional characterization of the cicatricial microbiota, including uncultivated representatives. Significant differences were found between the groups for 13 bacterial genera. In particular, in the rumen of cows treated with the probiotic Cellobacterin+, compared to the control group, a lower proportion of the order Clostridia were found, namely the bacteria of the genera Anaerofilum sp. (2.3 times lower, p ≤ 0.05) and Anaerostipes sp. (1.8 times lower, p ≤ 0.05) that produce lactate in the rumen as the end product of glucose metabolism. A decrease occurred in the abundance of the genera Campylobacter, Gemella, Mycoplasma, Shewanella (p ≤ 0.05), and Fusobacterium (including F. necrophorum) (p ≤ 0.001) among which pathogens are often found. Changes in the taxonomic structure of rumen microbiota as influenced by the probiotic were also associated with metabolic changes. The predicted functional potential of seven metabolic pathways was enhanced in cows fed Cellobacterin+ compared to the control animals. Thus, when fed Cellobacterin+, there was a 3.5-fold increase (p ≤ 0.05) in the predicted level of microbiome metabolic capabilities associated with the synthesis of glyoxylate from allantoin, and 2.3-fold increase (p ≤ 0.05) in the biosynthesis of propionate from L-glutamate. These findings allow us to suggest an important role of the biological product Cellobacterin+ for maintaining the homeostasis of metabolic processes.

Anomalous Phylogenetic Behavior of Ribosomal Proteins in Metagenome-Assembled Asgard Archaea.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process.

Ligation of 2', 3'-cyclic phosphate RNAs for the identification of microRNA binding sites.

Identifying the targetome of a microRNA is key for understanding its functions. Cross‐linking and immunoprecipitation (CLIP) methods capture native miRNA‐mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA‐mRNA sequences via ligation of 5′‐phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2′‐, 3′‐cyclic phosphate groups, as part of a new CLIP method that does not require 5′‐phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3′‐phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.

Analyses of DNA double-strand break repair pathways in tandem arrays of HXT genes of Saccharomyces cerevisiae.

Eukaryotic genomes contain numerous homologous repeat sequences including redundant genes with divergent homology that can be potential recombination targets. Recombination between divergent sequences is rare but poses a substantial threat to genome stability. The hexose transporter (HXT) gene family shares high sequence similarities at both protein and DNA levels, and some members are placed close together in tandem arrays. In this study, we show that spontaneous interstitial deletions occur at significantly high rates in HXT gene clusters, resulting in chimeric HXT sequences that contain a single junction point. We also observed that DNA double-strand breaks created between HXT genes produce primarily interstitial deletions, whereas internal cleavage of the HXT gene resulted in gene conversions as well as deletion products. Interestingly, interstitial deletions were less constrained by sequence divergence than gene conversion. Moreover, recombination-defective mutations differentially affected the survival frequency. Mutations that impair single-strand annealing (SSA) pathway greatly reduced the survival frequency by 10-1,000-fold, whereas disruption of Rad51-dependent homologous recombination exhibited only modest reduction. Our results indicate that recombination in the tandemly repeated HXT genes occurs primarily via SSA pathway.

Effects of Personal Exposure to Fine Particulate Matter Air Pollutionon Gut microbiotain elderly: The China BAPE study

Background: In recent years, it is confirmed that human gut microbiota is critical for human health. Animal studies suggest that air pollutants may alter the composition of the gut microbiota, which may negatively impact health through changes in the composition or function of the gut microbiome.However, Research on the effects of personal exposure to fine particulate matter on gut microbiota is lacking in developing countries, especially in highly polluted areas.Methods:Participants (60-69 years, n=76) were enrolled between Sep 10th 2018 and Jan 19th 2019 with a month interval.MicroPEMTM real-time personal measurements was used to monitor the subject's personal PM2.5 exposure. Faecal samples from 76 healthy elderly were sequenced using 16S rDNA amplification to assess microbial diversity, richness and relative taxa abundance. Operational Units were clustered with 97% similarity cutoff and chimeric sequences were identified and removed. A linear mixed-effect model was applied to estimate the associations of exposure to Fine Particulate Matter with alpha diversity indices and relative abundance of the gut microbiota. Results:A total of 516 genus level microbiota were identified in this study.The five most abundant bacteria in the fecal flora of the participants were Bacteroides, Prevotella, Pachyphyte, Faecalibacterium and Roseburia.Exposure to fine particulate matter was associated with the alterations in gut microbiota diversity and relative abundance of 20 genus level microbiota. By analyzing the abundance composition of the KEGG pathway of the intestinal flora of the Participants,the pathways enriched by the intestinal flora mainly include transport, DNA repair and recombinant proteins, ribosomes, and purine metabolism. Involves environmental information processing, genetic information processing, and metabolic mechanisms.Conclusions: Exposure to fine particulate matter may alter the relative abundance, and decrease the diversity of the gut microbiota.These findings can extend our understanding of the mechanism of air pollution inducing diseases.

DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences

Abstract There are few available tools to comprehensively and economically identify uncharacterized flanking regions that are not extremely labour intensive and which exploit the advantages of emerging long‐read sequencing platforms. We describe SIP; a sonication‐based inverse PCR high‐throughput sequencing strategy to investigate uncharacterized flanking region sequences, including those flanking mobile DNA. SIP combines unbiased fragmentation by sonication and target enrichment by coupling outward facing PCR priming with long‐read sequencing technologies. We demonstrate the effectiveness of SIP by determining retroviral integrations which are high copy and challenging to characterize. We further describe SIP's workflow, examine retroviral (proviral) enrichment and characterize viral structural variants identified. When SIP was coupled with long‐read sequencing using the PacBio RS II platform, proviral integration was extensively characterized at high sequence depth per integration. By interrogating the sequence data, we were also able to test several intrinsic factors including SIP's propensity to form chimeric sequences and adapter ligation efficiencies. SIP is an adaption of a traditional molecular biology technique that can be used to characterize any unknown genomic flanking sequence or to extend any sequence for which only minimal sequence information is available. SIP can be applied broadly to study complex biological systems such as mobile genetic elements with high throughput.

Effects of graphene oxide on PCR amplification for microbial community survey

BackgroundGraphene oxide (GO) has been suggested as an efficient assistant additive to eliminate non-specific amplification of the polymerase chain reaction (PCR). Although many studies have focused on exploring its molecular mechanism, the practice of GO on the quantitation of microbial community has not been implemented yet. In this study, GO was added in PCR system to explore the changes on removing typical amplification errors, such as chimera and mismatches on two kinds of mock communities (an evenly mixed and a staggered mock communities) and environmental samples.ResultsHigh-throughput sequencing of bacterial and fungal communities, based on 16S rRNA genes and internal transcribed spacers (ITS) respectively, showed that GO could significantly increase large segmental error (chimeric sequence) in PCR procedure while had no specific effect on point error (mismatched sequence). Besides, GO reduced the α-diversity of community, and changed the composition of fungal community more obviously than bacterial community.ConclusionsOur study provides the first quantitative data on microbial community level to prove the negative effect of GO, and also indicates that there may be a more complex interaction between GO and comprehensive DNA fragments in PCR process.

De novo sequence assembly requires bioinformatic checking of chimeric sequences.

De novo assembly of sequence reads from next generation sequencing platforms is a common strategy for detecting presence and sequencing of viruses in biospecimens. Amplification artifacts and presence of several related viruses in the same specimen can lead to assembly of erroneous, chimeric sequences. We now report that such chimeras can also occur between viral and non-viral biological sequences incorrectly joined together which may cause erroneous detection of viruses, highlighting the importance of performing a chimera checking step in bioinformatics pipelines. Using Illumina NextSeq and metagenomic sequencing, we analyzed 80 consecutive non-melanoma skin cancers (NMSCs) from 11 immunosuppressed patients together with 11 NMSCs from patients who had only developed 1 NMSC. We aligned high-quality reads against a Human Papillomavirus (HPV) database and found HPV sequences in 9/91 specimens. A previous bioinformatic analysis of the same crude sequencing data from some of these samples had found an additional 3 specimens to be HPV-positive after performing de novo assembly. The reason for the discrepancy was investigated and found to be mostly caused by chimeric sequences containing both viral and non-viral sequences. Non-viral sequences were present in these 3 samples. To avoid erroneous detection of HPV when performing sequencing, we thus developed a novel script to identify HPV chimeric sequences.