Single-molecule Real-time Sequencing Research Articles

Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1

Members of the genus Streptococcus inhabit a variety of sites in humans and other animals and some species are prolific producers of proteinaceous antibiotics (bacteriocins). As little is known about (i) streptococci inhabiting domestic pets, and (ii) whether novel bacteriocin-producing streptococci can be isolated from domestic pets, the aim of this study is to address these gaps in the research literature. In this study, Streptococcus equinus MDC1, isolated from a healthy dog, was found to exhibit potent antibacterial activity against Micrococcus luteus in a simultaneous antagonism assay, suggesting that strain MDC1 produces a lantibiotic bacteriocin. The inhibitory activity spectrum of S. equinus MDC1, determined using agar-based deferred antagonism assays against >70 indicator strains, was found to be similar to that of nisin U (a lantibiotic produced by Streptococcus uberis). However, the spectra of the two bacteriocins differed by 23 strains, mainly with the MDC1 bacteriocin having no inhibitory activity towards certain streptococci of human origin (e.g., Streptococcus gordonii, Streptococcus anginosus, Streptococcus salivarius). The genome of S. equinus MDC1, which was sequenced completely using single-molecule real-time (SMRT) next-generation DNA sequencing technology, comprises a single 1,936,555-basepair chromosome containing seven copies of the ribosomal RNA operon, 69 tRNA genes and nearly 1900 putative coding sequences. Analysis of the MDC1 genome sequence using the bacteriocin detection algorithms BAGEL4 and antiSMASH revealed the location of a 13,164-basepair 11-gene locus, designated nmd, which encoded a mature nisin E peptide that differed from nisin U by only two amino acids (Ile15→Ala and Leu21→Ile) and an extra C-terminal asparagine residue, and the proteins required for post-translational modification of the bacteriocin, processing, export, and producer immunity. Despite the high homology (90.6% identity, 93.8% similarity) between nisin E and nisin U, there was considerably less homology (47.4–76.3% identity, 68.4–88.8% similarity) between the other proteins encoded by their respective biosynthetic loci. This new natural variant of nisin, called nisin E, represents the first nisin variant to be reported for S. equinus; additionally, its differences with nisin U may provide some insight into the amino acids that influence bacteriocin potency and killing spectrum.

SMRT and Illumina sequencing provide insights into mechanisms of lignin and terpenoids biosynthesis in Pinus massoniana Lamb

Wood and oleoresin are important industrial raw materials with high economic value; however, their molecular formation and biosynthesis mechanisms in different tissues of Pinus massoniana remain unexplored. Therefore, we used single-molecule real-time sequencing technology (SMRT) and Illumina RNA sequencing to establish a transcriptome dataset and explore the expression pattern of genes related to secondary metabolites involved in wood formation and oleoresin biosynthesis in six different P. massoniana tissues. In total, 63.58 Gb of polymerase reads were obtained, including 41,407 isoforms with an average length of 1822 bp. We identified 3939 and 8785 isoforms and 161 and 481 transcription factors with tissue expression specificity and in the reproductive and vegetative organs, respectively. Eighty isoforms were annotated as cellulose synthases and 224 isoforms involved in lignin biosynthesis were enriched. Additionally, we identified 217 isoforms involved in the terpenoid biosynthesis pathway, with needles having the most tissue-specific genes for terpenoid biosynthesis. Some isoforms related to lignin biosynthesis were highly expressed in the xylem, according to the results of transcriptome sequencing and real-time quantitative reverse-transcription polymerase chain reaction. Our research confirmed the advantages of SMRT sequencing and provided valuable information for the transcriptional annotation of P. massoniana, which will be beneficial for producing better raw wood and oleoresin materials.

Microbial Diversity and Volatile Flavor Compounds in Tibetan Flavor Daqu.

In this study, the microbial diversity in Tibetan flavor Daqu was analyzed based on single molecule real-time sequencing (SMRT). The volatile flavor compounds in Daqu were detected using the headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). In addition, the correlation between various microbes and volatile flavor compounds was explored. Our studies indicated that the dominant bacterial genera in Tibetan flavor Daqu were Oceanobacillus, Kroppenstedtia, Virgibacillus, Enterococcus, Pediococcus, Streptomyces, Saccharopolyspora, Leuconostoc, uncultured_bacterium_f_Lachnospiraceae and Lactobacillus. The dominant fungal genera were Wickerhamomyces, Monascus, Aspergillus and Rhizomucor. 101 volatile compounds were detected in the Daqu samples, including alcohols, acids, esters, aldehydes, hydrocarbons, ketones, ethers, aromatics and pyrazines, and 10 key flavor compounds were identified using the relative odor activity value (ROAV). The results of our correlation analysis showed that Enterococcus was mainly associated with the synthesis of aldehydes such as trans-2-octenal, and uncultured_bacterium_f_lachnospiraceae was associated with the synthesis of most aldehydes. This paper has systematically investigated the physicochemical indices, microbial community structure and flavor compounds of Tibetan flavor Daqu, which is helpful in gaining a deeper understanding of the characteristics of Tibetan flavor Daqu.

Stabilization of jiuyao quality for huangjiu brewing by fortifying functional strains based on core microbial community analysis

Jiuyao as a generally small saccharifying and spontaneously fermenting starter is indispensable for huangjiu brewing. However, jiuyao currently in use is facing a major challenge of standardized manufacturing because of its open operating environment and rather primitive operations. This study provided a stead manufacturing procedure for jiuyao by controlling the processing parameters. Saccharomycopsis fibuligera and Rhizopus microsporus were revealed as the core functional species related to starch-degrading enzyme activities with single-molecule real-time (SMRT) sequencing. Two strains with high starch-degrading abilities were successfully isolated and identified as Saccharomycopsis fibuligera CY2111 and Rhizopus microsporus SM4. Meanwhile, the environmental fermentation parameters of jiuyao were optimized to enhance enzyme activities. The glucoamylase and α-amylase activities of fortified jiuyao with strain CY2111 and SM4 were 3.07 and 3.15 times that of traditional jiuyao, respectively. Moreover, the alcohol content increased by approximately 7.5% when fortified jiuyao was used for huangjiu brewing. The flavor profiles determined by gas chromatography–mass spectrometry indicated there was no significant difference in the key aromas between the huangjiu fermented with fortified jiuyao and that of traditional jiuyao. Further, fortified jiuyao produced more pleasant esters in huangjiu such as ethyl isovalerate, isoamyl acetate, and ethyl caprylate. Combined with the sensory evaluation, huangjiu fermented with fortified jiuyao presented stronger fruity aroma. These results demonstrated that careful fermentation settings combined with biofortification technology were feasible to improve jiuyao quality, thus promoting huangjiu flavor. Taken together, it provided scientific guidance to improve traditional handcrafting and scale up the production of jiuyao under controllable fermentation.

Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass.

Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, Clarireedia monteithiana and C. jacksonii (formerly Sclerotinia homoeocarpa) have been reported so far in the United States To study the Clarireedia genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced via Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as C. aff. paspali, a novel species in the United States Using short and long reads, C. aff. paspali H3 contained 193 contigs with 48.6Mbp and presented the most completed assembly and annotation among Clarireedia species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher Clarireedia pathogenicity, C. aff. paspali genomes (H2 and H3), as well as available C. jacksonii (LWC-10 and HRI11), C. monteithiana (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in S. sclerotiorum. Seventeen orthologs of pathogenicity genes have been identified in Clarireedia species involved in oxalic acid production (pac1, nox1), mitogen-activated protein kinase cascade (pka1, smk3, ste12), appressorium formation (caf1, pks13, ams2, rgb1, rhs1) and glycolytic pathway (gpd). Within these genes, 366 species-specific SNPs were recorded between Clarireedia species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among Clarireedia spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among Clarireedia species.

High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry.

Cultivated strawberry (Fragaria × ananassa), a perennial herb belonging to the family Rosaceae, is a complex octoploid with high heterozygosity at most loci. However, there is no research on the haplotype of the octoploid strawberry genome. Here we aimed to obtain a high-quality genome of the cultivated strawberry cultivar, "Yanli", using single molecule real-time sequencing and high-throughput chromosome conformation capture technology. The "Yanli" genome was 823Mb in size, with a long terminal repeat assembly index of 14.99. The genome was phased into two haplotypes, Hap1 (825Mb with contig N50 of 26.70Mb) and Hap2 (808Mb with contig N50 of 27.51Mb). Using the combination of Hap1 and Hap2, we obtained for the first time a haplotype-resolved genome with 56 chromosomes for the cultivated octoploid strawberry. We identified a ~ 10Mb inversion and translocation on chromosome 2-1. 104 957 and 102 356 protein-coding genes were annotated in Hap1 and Hap2, respectively. Analysis of the genes related to the anthocyanin biosynthesis pathway revealed the structural diversity and complexity in the expression of the alleles in the octoploid F. × ananassa genome. In summary, we obtained a high-quality haplotype-resolved genome assembly of F. × ananassa, which will provide the foundation for investigating gene function and evolution of the genome of cultivated octoploid strawberry.

Structure and Alternative Splicing of the Antisense FMR1 (ASFMR1) Gene.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by an expansion of 55-200 CGG repeats (premutation) in the 5'-UTR of the FMR1 gene. Bidirectional transcription at FMR1 locus has been demonstrated and specific alternative splicing of the Antisense FMR1 (ASFMR1) gene has been proposed to have a contributing role in the pathogenesis of FXTAS. The structure of ASFMR1 gene is still uncharacterized and it is currently unknown how many isoforms of the gene are expressed and at what level in premutation carriers (PM) and if they may contribute to the premutation pathology. In this study, we characterized the ASFMR1 gene structure and the transcriptional landscape by using PacBio SMRT sequencing with target enrichment (IDT customized probe panel). We identified 45 ASFMR1 isoforms ranging in sizes from 523bp to 6Kb, spanning approximately 59kb of genomic DNA. Multiplexing and sequencing of six human brain samples from PM samples and normal control (HC) were carried out on the PacBio Sequel platform. We validated the presence of these isoforms by qRT-PCR and Sanger sequencing and characterized the acceptor and donor splicing site consensus sequences. Consistent with previous studies conducted in other tissue types, we found a high expression of ASFMR1 isoform Iso131bp in brain samples of PM as compared to HC, while no differences in expression levels were observed for the newly identified isoforms IsoAS1 and IsoAS2. We investigated the role of the splicing regulatory protein Sam68 which we did not observe in the alternative splicing of the ASFMR1 gene. Our study provides a useful insight into the structure of ASFMR1 gene and transcriptional landscape along with the expression pattern of various newly identified novel isoforms and on their potential role in premutation pathology.

SMRT sequencing and characterization of putative hydroxycinnamoyl-CoA: Quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes responsible for biosynthesis of structurally diverse chlorogenic acids in Bidens pilosa

Bidens pilosa L. is an underutilised plant that serves as an ingredient for traditional herbal medicine and as food. This plant has received considerable attention because of its chemistry, which comprises of chemicals from various classes, with those from the phenylpropanoid pathway (e.g., chlorogenic acids) existing in abundance. B. pilosa L. produces chemically diverse chlorogenic acids (CGAs), characterised mainly by isomers thereof. The enzymes involved in the biosynthesis of chlorogenic acids have been decoded and characterised in many plants. However, genes that play a role in the biosynthesis of chlorogenic acids in B. pilosa L. are not yet described and, as such, the aim of the current study was to identify different isoforms of the Hydroxycinnamoyl-CoA: quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes that play a role in the diversification of chlorogenic acids in Bidens pilosa L. Herein, a robust gene sequencing technology through Single Molecule Real Time (SMRT) approach was applied to establish the gene sequences encoding transferases responsible for CGA production in B. pilosa L. Sequence homology of the established genes was evaluated by means of multiple sequence alignment against already published orthologous genes from closely related plants. Thereafter, phylogeny trees were constructed to graphically display the taxonomical relationship with other chlorogenic acid producing plants. To further demonstrate the functionality of these genes in plants, chlorogenic acid profiling was carried out using Liquid chromatography quadrupole time of flight mass spectrometer (LC-QTOF-MS). B. pilosa L. plants produced isomeric forms of mono, di and tri-acylated CGAs. A total of three HQT genes and one HCT gene have been identified in this study and have been shown through homology to be related with other related genes from other CGAs producing plants. Future work should involve expression of the identified genes in other non-chlorogenic acid producing plants to enhance their nutraceutical value.

Long-read sequencing improves diagnostic rate in neuromuscular disorders.

Massive parallel sequencing methods, such as exome, genome, and targeted DNA sequencing, have aided molecular diagnosis of genetic diseases in the last 20 years. However, short-read sequencing methods still have several limitations, such inaccurate genome assembly, the inability to detect large structural variants, and variants located in hard-to-sequence regions like highly repetitive areas. The recently emerged PacBio single-molecule real-time (SMRT) and Oxford nanopore technology (ONT) long-read sequencing (LRS) methods have been shown to overcome most of these technical issues, leading to an increase in diagnostic rate. LRS methods are contributing to the detection of repeat expansions in novel disease-causing genes (e.g., ABCD3, NOTCH2NLC and RILPL1 causing an Oculopharyngodistal myopathy or PLIN4 causing a Myopathy with rimmed ubiquitin-positive autophagic vacuolation), of structural variants (e.g., in DMD), and of single nucleotide variants in repetitive regions (TTN and NEB). Moreover, these methods have simplified the characterization of the D4Z4 repeats in DUX4, facilitating the diagnosis of Facioscapulohumeral muscular dystrophy (FSHD). We review recent studies that have used either ONT or PacBio SMRT sequencing methods and discuss different types of variants that have been detected using these approaches in individuals with neuromuscular disorders.

Characterization and Analysis of the Full-Length Transcriptome Provide Insights into Fruit Quality Formation in Kiwifruit Cultivar Actinidia arguta cv. Qinziyu

Kiwifruit is an economically important horticultural crop with extremely high values in nutrition and health care. However, the molecular mechanisms underlying fruit quality formation remain largely limited for most kiwifruit varieties. Recently, a new kiwifruit cultivar with a high level of soluble solids, Actinidia arguta cv. Qinziyu (full-red flesh) was discovered through the introduction and propagation test. To provide new insights into fruit quality formation in a typical kiwifruit cultivar, we integrated full-length transcriptome surveys based on PacBio single-molecule real-time (SMRT) sequencing, key enzyme genes expression involved in carbohydrate and amino acids metabolism pathways, and bHLH gene family analysis to enhance the understanding of soluble sugar, organic acid, and anthocyanin biosynthesis in A. arguta cv. Qinziyu. A total of 175,913 CCSs were generated, of which 124,789 were identified as FLNC transcripts. In total, 45,923 (86.99%) transcripts were successfully annotated, and more than 76.05% of the transcripts were longer than 1 Kb. KEGG pathway analysis showed that 630 candidate genes encoding 55 enzymes were mainly involved in carbohydrate and amino acid biosynthesis pathways. Further analysis verified the expression of 12 key enzyme genes (e.g., pyruvate kinase (PK), enolase (ENO), hexokinase (HK), and phosphoglycerate kinase (PGK)) in flowers using quantitative real-time PCR. Furthermore, we also screened 10 AabHLH proteins’ function in anthocyanin biosynthesis and characterized the AabHLH gene family in A. arguta cv. Qinziyu. Overall, our research data generated by SMRT technology provide the first set of gene isoforms from a full-length transcriptome in A. arguta cv. Qinziyu and more comprehensive insights into the molecular mechanism of fruit quality formation.

Detection of DNA Modification Using Nanopore Sequencers.

DNA modification is a crucial factor of epigenetic modification and has vital functions for gene regulation and phenotype control. A profound understanding of DNA modification requires precise mapping of the modified bases on genomic DNA. In addition to methods such as bisulfite sequencing and single-molecule real-time (SMRT) sequencing of PacBio sequencers, nanopore sequencers can be also utilized for the detection of DNA modification. Here, I will briefly review the three methods for the detection of DNA modification with nanopore sequencers and introduce a protocol using MinION and Megalodon.

Crossing Bacterial Genomic Features and Methylation Patterns with MeStudio: An Epigenomic Analysis Tool.

DNA methylation is one of the most observed epigenetic modifications. It is present in eukaryotes and prokaryotes and is related to several biological phenomena, including gene flow and adaptation to environmental conditions. The widespread use of third-generation sequencing technologies allows direct and easy detection of genome-wide methylation profiles, offering increasing opportunities to understand and exploit the epigenomic landscape of individuals and populations. Here, we present a pipeline named MeStudio, with the aim of analyzing and combining genome-wide methylation profiles with genomic features. Outputs report the presence of DNA methylation in coding sequences (CDSs) and noncoding sequences, including both intergenic sequences and sequences upstream of the CDS. We apply this novel tool, showing the usage and performance of MeStudio, on a set of single-molecule real-time sequencing outputs from strains of the bacterial species Sinorhizobium meliloti.

A survey of the full-length transcriptome of Gracilariopsis lemaneiformis using single-molecule long-read sequencing

BackgroundPosttranscriptional processing of precursor mRNAs contributes to transcriptome and protein diversity and gene regulatory mechanisms in eukaryotes. However, this posttranscriptional mechanism has not been studied in the marine macroalgae Gracilariopsis lemaneiformis, which is the most cultivated red seaweed species in China.ResultsIn the present study, third-generation sequencing (Pacific Biosciences single-molecule real-time long-read sequencing, SMRT-Seq) was used to sequence the full-length transcriptome of G. lemaneiformis to identify alternatively spliced transcripts and alternative polyadenylation (APA) sites in this species. RNAs were isolated from G. lemaneiformis under various treatments including abiotic stresses and exogenous phytohormones, and then equally pooled for SMRT-Seq. In summary, 346,544 full-length nonchimeric reads were generated, from which 13,630 unique full-length transcripts were obtained in G. lemaneiformis. Compared with the known splicing events in the gene models, more than 3000 new alternative splicing (AS) events were identified in the SMRT-Seq reads. Additionally, 810 genes were found to have poly (A) sites and 91 microRNAs (miRNAs), 961 long noncoding RNAs and 1721 novel genes were identified in G. lemaneiformis. Moreover, validation experiments showed that abiotic stresses and phytohormones could induce some specific AS events, especially intron retain isoforms, cause some alterations to the relative ratios of transcripts annotated to the same gene, and generate novel 3′ ends because of differential APA. The growth of G. lemaneiformis was inhibited by Cu stress, while this inhibition was alleviated by ACC treatment. RNA-Seq analysis further revealed that 211 differential alternative splicing (DAS) events and 142 DAS events was obtained in CK vs Cu and Cu vs Cu + ACC, respectively, suggesting that AS of functional genes could be regulated by Cu stress and ACC. Compared with Cu stress, the expression of transcripts with DAS events mainly involved in the carbon fixation in photosynthetic organisms and oxidative phosphorylation pathway was upregulated in Cu + ACC treatment, revealing that ACC alleviated the growth inhibition by Cu stress by increasing carbon fixation and oxidative phosphorylation.ConclusionsOur results provide the first comprehensive picture of the full-length transcriptome and posttranscriptional mechanism in red macroalgae, including transcripts that appeared in the presence of common abiotic stresses and phytohormones, which will improve the gene annotations of Gracilariopsis and contribute to the study of gene regulation in this important cultivated seaweed.

Single-Molecule Real-Time Sequencing of Full-Length Transcriptome and Identification of Genes Related to Male Development in Cannabis sativa.

Female Cannabis sativa plants have important therapeutic properties. The sex ratio of the dioecious cannabis is approximately 1:1. Cultivating homozygous female plants by inducing female plants to produce male flowers is of great practical significance. However, the mechanism underlying cannabis male development remains unclear. In this study, single-molecule real-time (SMRT) sequencing was performed using a mixed sample of female and induced male flowers from the ZYZM1 cannabis variety. A total of 15,241 consensus reads were identified, and 13,657 transcripts were annotated across seven public databases. A total of 48 lncRNAs with an average length of 986.54 bp were identified. In total, 8202 transcripts were annotated as transcription factors, the most common of which were bHLH transcription factors. Moreover, tissue-specific expression pattern analysis showed that 13 MADS transcription factors were highly expressed in male flowers. Furthermore, 232 reads of novel genes were predicted and enriched in lipid metabolism, and qRT-PCR results showed that CER1 may be involved in the development of cannabis male flowers. In addition, 1170 AS events were detected, and two AS events were further validated. Taken together, these results may improve our understanding of the complexity of full-length cannabis transcripts and provide a basis for understanding the molecular mechanism of cannabis male development.

Transcriptome sequencing of the spleen of the Yangtze sturgeon (Acipenser dabryanus) under Edwardsiella tarda and poly(I:C) treatments

Acipenser dabryanus is an ancient living fossil and a flagship species of the Yangtze River. However, A. dabryanus was listed as a critically endangered species due to the dramatic decrease in its population, and disease outbreaks occurring during captive breeding have hindered the protection of this species. Here, we investigated the effect of the pathogenic bacterium Edwardsiella tarda and poly(I:C) on A. dabryanus at the histopathological and transcriptomic levels. After 48 h of infection with E. tarda, A. dabryanus exhibited marked pathological changes in the intestine and liver. We then conducted full-length transcriptome sequencing of the spleen by SMRT sequencing technology. Using the full-length transcriptome data as a reference, transcriptomic results obtained through Illumina sequencing technology revealed that a total of 894 and 963 unigenes were differentially expressed between the PBS and bacterial infection groups and between the PBS and poly(I:C) challenge groups, respectively. E3 ubiquitin ligases family members, T-cell receptor and clusters of differentiation were significantly upregulated by bacteria or poly(I:C) treatment, while the complement was significantly downregulated, suggesting that they might participate in multiple immune pathways in the response to pathogen invasion. Pathogen infection also induced changes in some key rate-limiting enzymes involved in energy metabolism. Our study can provide important information on the molecular mechanisms underlying the immune response of fish to pathogenic microbial infections and help explore the antibacterial and antiviral immunity of A. dabryanus.

Transcriptome Analysis of the Differentially Expressed Heat-resistant Genes between Calliptamus italicus and Gomphocerus sibiricus.

Calliptamus italicus and Gomphocerus sibiricus are indicator species in Xinjiang's low-altitude (700-1,900 m) and high-altitude (2,000-3,400 m) grasslands, respectively. C. italicus is tolerant to high-temperature stress, with its semilethal temperature (LT50) being 10.5°C higher than that of G. sibiricus. The two locust species were subjected to high-temperature stress to explore the molecular mechanisms and differences in high temperature tolerance between the two locust species. Next, the next generation sequencing (NGS) data were mapped to reference transcripts obtained using single molecule real Time (SMRT) sequencing to construct a nonparameter transcriptome. The transcriptomic response of these two locust species displayed different patterns. C. italicus had 126 differentially expressed genes (DEGs), with 59 and 67 being significantly up-regulated and down-regulated, respectively. The heat shock protein (Hsp) genes were highly expressed upon two locust species exposure to high-temperature stress, with Hsp70 being expressed the most. G. sibiricus had 86 DEGs, of which 45 were significantly up-regulated and 41 significantly down-regulated. In addition, the expression of the key enzyme encoding gene Myo-inositol oxygenase (MIOX) in inositol degradation was the highest in G. sibiricus. In the KEGG pathway, the biological processes and metabolic pathways were the most enriched pathways in C. italicus and G. sibiricus, respectively. Moreover, the quantitative fluorescence results were consistent with the transcriptome results, implying that the transcriptome results were accurate. The findings in this study provide valuable information for future research exploring the evolution mechanisms of heat resistance in C. italicus and G. sibiricus.

Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Genome Resource for Acinetobacter schindleri H4-3-C1: An Endophyte of Pseudostellaria heterophylla with Degradation Activity to Toxins Produced by Fungal Pathogens

Acinetobacter schindleri is an endophyte of Pseudostellaria heterophylla, a traditional Chinese herbal plant. It has high degradation activity to toxins produced by fungal pathogen Fusarium graminearum. Here, we deployed PacBio single-molecule real-time long-read sequencing technology to generate a complete genome assembly for the Acinetobacter schindleri H4-3-C1 strain and obtained 1.59 Gb of clean reads. These reads were assembled to a single circular DNA chromosome with a length of 3,265,024 bp, and no plasmid was found in the genome. Totals of 3,193 coding sequences, 91 transfer RNA, 21 ribosomal RNA, and 75 small RNAs were identified in the genome. This high-quality genome assembly and gene annotation resource will facilitate the excavation of the zearalenone degradation gene and provide valuable resources for preventing and controlling toxigenic fungal diseases of P. heterophylla. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Tandem repeats structure of gel-forming mucin domains could be revealed by SMRT sequencing data

Mucins are large glycoproteins that cover and protect epithelial surface of the body. Mucin domains of gel-forming mucins are rich in proline, threonine, and serine that are heavily glycosylated. These domains show great complexity with tandem repeats, thus make it difficult to study the sequences. With the coming of single molecule real-time (SMRT) sequencing technologies, we manage to present sequence structure of mucin domains via SMRT long reads for gel-forming mucins MUC2, MUC5AC, MUC5B and MUC6. Our study shows that for different individuals, single nucleotide polymorphisms could be found in mucin domains of MUC2, MUC5AC, MUC5B and MUC6, while different number of tandem repeats could be found in mucin domains of MUC2 and MUC6. Furthermore, we get the sequence of MUC2, MUC5AC, and MUC5B mucin domain in a Chinese individual for each nucleotide at accuracy of possibly 99.98–99.99%, 99.93–99.99%, and 99.76–99.99%, respectively. We report a new method to obtain DNA sequence of gel-forming mucin domains. This method will provided new insights on getting the sequence for Tandem Repeat parts which locate in coding region. With the sequences we obtained through this method, we can give more information for people to study the sequences of gel-forming mucin domains.