Illumina Paired-end Sequencing Research Articles

Draft Genome Sequence of Lactobacillus oris BE7N, Isolated from a Healthy Centenarian in Hainan, China.

We present the draft genome sequence of Lactobacillus oris strain BE7N, which was isolated from a healthy male centenarian in Hainan, China. The final 2,129,000-bp draft genome has a G+C content of 50.05%. The genome was sequenced using paired-end Illumina sequencing.

Development of twenty-one novel microsatellite loci for Gila topminnow, Poeciliopsis occidentalis occidentalis.

Gila topminnow (Poeciliopsis occidentalis occidentalis) was once highly abundant throughout the Lower Colorado River Basin of the southwestern United States. However, this Sonoran Desert endemic suffered extreme population declines over the past century because of habitat degradation and nonnative species introductions. Much of the prior conservation genetic work conducted on the species relied upon a small number of microsatellite loci; many exhibiting low variability in extant populations. Consequently, there was a need for additional microsatellite loci to provide high-resolution delimitation of populations for conservation purposes. Paired-end Illumina sequencing was utilized to screen the Gila topminnow genome for novel microsatellite loci. We identified 21 novel loci that exhibited no deviations from expectations of genetic equilibrium, and cross-amplified in Yaqui topminnow (P. o. sonoriensis). These loci were amplified from 401 samples representing eight populations of Gila topminnow and Yaqui topminnow. Although diversity was low for all populations (observed heterozygosity = 0.12 to 0.45), these novel markers provided ample power to identify population of origin for each individual in Bayesian assignment tests. This novel set of microsatellite loci provide a useful genetic tool to assess population genetic parameters of the endangered Gila topminnow and delineate populations for identifying conservation priorities. The cross-amplification of these loci in Yaqui topminnow shows promise for application to other Poeciliopsis species of Mexico and Central America.

Transcriptome sequencing of sesame (Sesamum indicum) using Illumina Platform

Sesame is an important oil seed crop worldwide and has essential health and medicinal values. In the present study, ahigh-throughput transcriptome sequencing of sesame was performed using Illumina paired-end sequencing technologyfor gene and marker discovery. Approximately 6 Gb data was generated and assembled into 16548 unigenes withan N50 of 905 bp. In addition, a total of 1716 unigeneswere assigned to 22 KEGG pathways. The unigenes resultedfrom this study are involved in lipid metabolism and Glycan biosynthesis pathways etc. Furthermore, 1443 SimpleSequence Repeats (SSR) were detected and consequently primers were designed.Our study enhances the genomic resources of sesame and provides ample amount of information about the transcriptome and SSRs which could serveas a valuable basis for future studies.

Weak range-wide population structure in the blackfin tuna (Thunnus atlanticus) revealed by analysis of genome-wide SNPs

Abstract Blackfin tuna (Thunnus atlanticus) is a small tuna distributed in the western Atlantic Ocean where it is exploited by growing recreational and commercial regional fisheries. In this work, genome-wide genetic variation was analysed to investigate the occurrence of stock subdivision. A de novo assembly of the blackfin tuna genome was generated using Illumina paired-end sequencing data and applied as a reference for population genomic analysis of specimens from nine localities (average sample size per locality n = 72) spanning most of the blackfin tuna distribution range. A total of 2139 single-nucleotide polymorphisms were discovered and genotyped using the double-digest restriction associated DNA sequencing. Pairwise exact homogeneity tests were significant in 24 out of 36 population pairs and significant spatial autocorrelation of genotypes was observed for specimens collected within 2250 km of each other. However, divergence among locality samples was very low (pairwise FST range 0.0002–0.0025) and significant temporal variations were detected in localities sampled multiple times. Approaches to detect cryptic groups de novo were unsuccessful. Additional sampling is warranted to determine if multiple stocks need to be defined for management and assess temporal and spatial patterns of gene flow connecting them.

The complete chloroplast genome sequence of Castanopsis fordii Hance (Fagaceae)

Castanopsis fordii Hance 1884 is a typical evergreen broad-leaved forest plant in the south subtropical and middle subtropical regions of China. It has high utilization value in wood production and soil erosion protection. Here, we first reported and characterized the complete chloroplast (cp) genome sequence of C. fordii based on Illumina paired-end sequencing data. The complete cp genome sequence of C. fordii was 160,853 base pairs (bp) in length which contained two inverted repeats (IRs) of 25,699 bp separated by a large single-copy (LSC) and a small single copy (SSC) of 90,474 bp and 18,981 bp, respectively. The cpDNA contained 129 genes, comprising 85 protein-coding genes, 36 tRNA genes, 8 rRNA genes. The overall GC content of the plastome was 36.8%. Phylogenetic analysis base on 14 chloroplast genomes indicated that C. fordii was closely related to the species C. tibetana and C. concinna in Fagaceae.

Genome-wide characterization leading to simple sequence repeat (SSR) markers development in Shorea robusta.

Tropical rainforests in Southeast Asia are enriched by multifarious biota dominated by Dipterocarpaceae.In this family, Shorea robusta is an ecologically sensitive and economically important timber species whose genomic diversity and phylogeny remain understudied due to lack of datasets on genetic resources. Smattering availability of molecular markers impedes population genetic studies indicating a necessity to develop genomic databases and species-specific markers in S. robusta. Accordingly, the present study focused on fostering de novo low-depth genome sequencing, identification of reliable microsatellites markers, and their validation in various populations of S. robusta in Uttarakhand Himalayas. With 69.88 million raw reads assembled into 1,97,489 contigs (read mapped to 93.2%) and a genome size of 357.11Mb (29 × coverage), Illumina paired-end sequencing technology arranged a library of sequence data of ~ 10 gigabases (Gb). From 57,702 microsatellite repeats, a total of 35,049 simple sequence repeat (SSR) primer pairs were developed. Afterward, among randomly selected 60 primer pairs, 50 showed successful amplification and 24 were found aspolymorphic. Out of which, nine polymorphicloci were further used for genetic analysis in 16 genotypes each from three different geographical locations of Uttarakhand (India). Prominently, the average number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.44, 0.324, 0.277 and 0.252, respectively. The accessibility of sequence information and novel SSR markers potentially enriches the current knowledge of the genomic background for S. robusta and to be utilized in various genetic studies in speciesunder tribe Shoreae.

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.

The complete chloroplast genome sequence of Berchemia racemosa Siebold & Zucc. (Rhamnaceae), a rare plant species in Korea

Berchemia racemosa Siebold & Zucc., 1845 is a rare species distributed in restricted areas in the western Korean peninsula. In this study, the complete chloroplast genome (plastome) of B. racemosa was sequenced and assembled by Illumina paired-end sequencing. The plastome of B. racemosa was 161,187 bp in length and was quadripartite in structure, including a large single-copy (LSC) region of 89,503 bp, a small single-copy (SSC) region of 18,214 bp, and two inverted repeats of 26,735 bp. The GC content was 37.2%. The plastome of B. racemosa contains 130 genes, including eight ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes, and 85 protein-coding genes. Phylogenetic analysis using complete genome sequences showed that B. racemosa is most closely related to Berchemia flavescens.

Characterization of the complete chloroplast genome of Firmiana hainanensis (Malvaceae), an endemic and vulnerable tree species of China

Firmiana hainanensis Kosterm. is a commercially valuable endemic tree species in China and has long been considered a globally vulnerable species. We assembled and characterized the complete chloroplast genome of this species by using Illumina pair-end sequencing data. The total chloroplast genome size was 161,559 bp, including two inverted repeats (IRs) of 25,612 bp, separated by a large single copy (LSC) and a small single copy (SSC) regions of 90,057 and 20,277 bp, respectively. A total of 130 genes were identified, including 85 protein-coding genes, 37 tRNA, and eight rRNA genes. Phylogenetic analysis showed that F. hainanensis was the most basal species in the genus Firmiana. The chloroplast genome of this species will provide a theoretical basis to understand the taxa’s evolution further and is expected to contribute to its conservation efforts.

Abstract A011: Whole genome sequencing of the Alabama Hereditary Cancer Cohort to identify African American breast cancer genetic risk variants

Abstract Regardless of having higher rates of triple-negative breast cancer (TNBC) and breast cancer-related deaths, African Americans are drastically understudied in hereditary breast cancer research. Our group has established the Alabama Hereditary Cancer Cohort to carry out next-generation sequencing (NGS) to address this gap. These efforts aim to identify inherited breast cancer risk variants in African Americans. Interestingly, African American TNBC tumors have been investigated through NGS, identifying somatic mutations unique to African American breast cancer. Now, African American-specific variants associated with inherited risk need to be identified, including those in genes harboring somatic mutations in African American TNBC. This study carried out whole genome sequencing (WGS) to investigate inherited African American breast cancer risk. All coding and non-coding regions of the nuclear genome and the entirety of the mitochondrial genome were explored, all of which can play a role in hereditary breast cancer risk. Individuals provided a blood or saliva sample for DNA extraction upon study consent. Subsequently, paired-end Illumina sequencing was carried out. Fastq files were trimmed using Trimmomatic and further processed according to the Genome Analysis ToolKit Best Practices, resulting in variant call format (VCF) files. Coding, non-coding, and mitochondrial regions were individually extracted using VCFtools and UCSC Table Browser tracks. Coding and mitochondrial variants were annotated using Annovar and MSeqDR, respectively. Coding variants were further filtered for minor allele frequency (MAF) classes (i.e., rare and low-frequency) and mutation type (i.e., protein-truncating variants; PTVs) using R (v. 4.2). Preliminary WGS data of 60 African American hereditary breast cancer cases have identified statistically significant rare and low-frequency PTVs in genes reported to harbor somatic mutations exclusively in African American TNBC tumors. Furthermore, we have identified variants in prostate cancer susceptibility genes that are statistically associated with African American breast cancer risk. We have also identified multiple variants in the mitochondrial genome that are statistically linked to breast cancer risk, some African American-specific. Our data shows that WGS is an invaluable tool for identifying breast cancer susceptibility variants. Further sequencing and functional analyses will validate our preliminary findings. Citation Format: Troy LoBue, Isaac Mcneely, Nancy Merner. Whole genome sequencing of the Alabama Hereditary Cancer Cohort to identify African American breast cancer genetic risk variants [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A011.

Climate-smart agricultural practices influence the fungal communities and soil properties under major agri-food systems.

Fungal communities in agricultural soils are assumed to be affected by climate, weather, and anthropogenic activities, and magnitude of their effect depends on the agricultural activities. Therefore, a study was conducted to investigate the impact of the portfolio of management practices on fungal communities and soil physical-chemical properties. The study comprised different climate-smart agriculture (CSA)-based management scenarios (Sc) established on the principles of conservation agriculture (CA), namely, ScI is conventional tillage-based rice-wheat rotation, ScII is partial CA-based rice-wheat-mungbean, ScIII is partial CSA-based rice-wheat-mungbean, ScIV is partial CSA-based maize-wheat-mungbean, and ScV and ScVI are CSA-based scenarios and similar to ScIII and ScIV, respectively, except for fertigation method. All the scenarios were flood irrigated except the ScV and ScVI where water and nitrogen were given through subsurface drip irrigation. Soils of these scenarios were collected from 0 to 15 cm depth and analyzed by Illumina paired-end sequencing of Internal Transcribed Spacer regions (ITS1 and ITS2) for the study of fungal community composition. Analysis of 5 million processed sequences showed a higher Shannon diversity index of 1.47 times and a Simpson index of 1.12 times in maize-based CSA scenarios (ScIV and ScVI) compared with rice-based CSA scenarios (ScIII and ScV). Seven phyla were present in all the scenarios, where Ascomycota was the most abundant phyla and it was followed by Basidiomycota and Zygomycota. Ascomycota was found more abundant in rice-based CSA scenarios as compared to maize-based CSA scenarios. Soil organic carbon and nitrogen were found to be 1.62 and 1.25 times higher in CSA scenarios compared with other scenarios. Bulk density was found highest in farmers' practice (Sc1); however, mean weight diameter and water-stable aggregates were found lowest in ScI. Soil physical, chemical, and biological properties were found better under CSA-based practices, which also increased the wheat grain yield by 12.5% and system yield by 18.8%. These results indicate that bundling/layering of smart agricultural practices over farmers' practices has tremendous effects on soil properties, and hence play an important role in sustaining soil quality/health.

Activity Evaluation of Eucommia ulmoides Extracts for Urinary System Disease Treatment and Phylogenetic Analysis

Eucommia ulmoides is a significant conventionalmedicinal plant in China, and is a prevalent herb for curing disease. Eucommia ulmoides is a significant medicinal plant for treating the urinary system disease treatment. After the extraction of active ingredient from Eucommia ulmoides, its application values on the urinary system disease were measured and the associated mechanism was also explored. At first, the ELISA was exploited for detecting of the inflammatory cytokines concentration that released into urine. Then, the pathological damage degree of kidney was also evaluated through HE staining after indicated treatment. Besides, for the Eucommia ulmoides, its complete chloroplast genome was analyzed through Illumina pair-end sequencing. According to neighbor-joining (NJ) phylogenetic analysis, the Lactuca sativa and E. ulmoides come together and become the sisters of other associated species.

Application value of Fagopyrum tataricum extracts in postpartum depression and phylogenetic analysis

Fagopyrum tataricum combines seven nutrients in one body. It is neither a medicine, nor a health care product, but a food that can part of a meal. F. tataricum has drawn the attention of most researchers because of the pharmacological activity of plant extracts. After the extraction of new compound from Fagopyrum tataricum, its preventive effect combined with psychological intervention on postpartum depression was evaluated, and the related mechanism was explored. The 5-HT content in the brain was first measured by ELISA. The relative expression of the 5-HT receptor on the neurons was determined with real-time RT-PCR assay. The complete chloroplast genome sequence of Fagopyrum tataricum was characterized through Illumina pair-end sequencing, and the neighbor-joining phylogenetic analysis of the F. tataricum was conducted.

Molecular Response Analysis By High Throughput Sequencing in Higher Risk Myelodysplastic Syndrome (HR-MDS) Treated Intensively with CPX-351

Context and objective Intensive chemotherapy (IC) remains an important therapeutic option in HR-MDS, but in-depth evaluation of response to IC remains ill studied. We previously reported results of CPX-351 in 31 untreated higher risk MDS patients (pts) (GFM-CPX-MDS study, NCT04273802, ASH 2021 Abst #243). Responses were associated with a significant decrease of CD34/CD117 progenitors and restoration of normal hematopoiesis patterns in bone marrow (BM) samples in multiparameter flow cytometry. In this ancillary study, the aim was to evaluate molecular responses by high-throughput sequencing (HTS) centrally. This involved description of the pts molecular landscape and an assessment of variant allele frequency (VAF) variations after induction treatment with CPX-351. Methods The mutational landscape was explored in BM nuclear cells by capture HTS of 42 genes (SureSelect XTHS2 Agilent) with unique molecular identifiers (UMI), unique dual index (UDI) and Illumina paired-end sequencing (Miseq and Nextseq). Somatic mutations (SM) were considered significant for VAFs >2%. Molecular response was evaluated by comparing baseline and post-induction. Molecular clearance was defined according to (i) clearance (<2%) of all SM excluding the "pre-leukemic” SM of DNMTA, TET2, ASXL1 (DTA) (ii) clearance of all non-DTA SM < HTS detection threshold (iii) normalized VAF decrease of the major non-DTA baseline SM (nmVAF). Results Median age of the 31 pts included was 62 years (range 31-69). There were 26 MDS-EB2 and 5 CMML-2. IPSS was int-2 in 26 pts and high in 5. Cytogenetic IPSS-R was very good (6.5%), good (71%, including normal in 22 pts), int (9.5%), poor (6.5%) or very poor (6.5%). After induction with CPX-351 (3 days cycle and a possible 2-day second induction cycle for non-responders), 16 pts (52%) reached CR, 4 (13%) CRi, 7 (22%) MLFS while 4 (13%) were in stable disease according to ELN 2017 criteria (which appeared more adequate than MDS IWG 2006, as reported at ASH 2021, Abst #243). Median follow-up was 511 days (179-710), 29/31 patients (94%) received allogeneic hematopoietic stem cell transplantation (Allo-HSCT) after, no, 1, 2, 3 or 4 CPX-351 consolidation cycles (that included only 1 day of CPX-351) in 10, 16, 7, 3 and 1 patients respectively. So far, 4 relapses have occurred, including 3 in transplanted pts. At inclusion, all pts had at least one mutation (median 3, [1-14]) with a mean VAF of 30% (2-89), implicating at least one DTA (71%; 22/31) or one non-DTA genes (93%; 29/31) (fig). Very high or high risk was seen in 19/31 (61%) pts according to the new IPSSm classification. Post induction molecular response was evaluated at a median of 53 days from CPX-351 onset (28-112) in 26 of the 27 who reached at least MLFS, and had a post-induction sample. Molecular clearance was observed (i) at the 2% threshold of non-DTA genes in 17/26 (65%) pts (ii) at the HTS detection threshold (0.1% i.e. minimum considered 3X with median coverage > 3000X) in 11/26 pts (42%) (iii) by nmVAF decrease in 25/26 pts (96%) with a median of -62% (-2 to -100%) (fig) while the remaining pt had an increase of SRSF2 (+67%) and RUNX1 (+46%) SMs but disappearance of three STAG2 SMs and was still in CR after allogeneic SCT. Of note, DTA SMs remained detectable in all pts with a median nmVAF modulation of -86% (+83% to -99.6%, fig) from baseline to post-induction. Of the 4 non-responders to CPX-351, 2 had high risk anomalies (inv(3) and EZH2 / RUNX1 SMs). The 3 relapses observed after alloHSCT, at 7, 8 and 15 months had nmVAFs decrease from baseline to post-induction of -95%, -98% and -50% respectively. Among them, one had a very complex karyotype and a homozygous TP53 mutation, the two others had a high mutational load and EZH2 mutations and one of the latest was transplanted in refractory state. Clonal evolution was observed in 5/26 pts with significant emergence of new mutations involving PPM1D, ASXL1 or JAK2 and in one pt the emergence of EZH2, RUNX1 and TET2 SMs. Four of these pts remained in morphological CR while one progressed to AML. Conclusion In 25/26 CPX-351 responders, a significant decrease of nmVAF was observed, confirming the interest of CPX-351 treatment in higher risk MDS, especially as a bridge to transplant. Follow up is ongoing and few relapses were seen to evaluate the predictive role of molecular analysis. An update will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Complete Genome Sequence of a Luteibacter Strain Isolated from a Carnivorous Pitcher Plant (Sarracenia minor).

A bacterial isolate of Luteibacter anthropi, designated SM7.4, was isolated from decaying insect detritus inside a carnivorous pitcher plant (Sarracenia minor). Here, we report a complete genome sequence for Luteibacter anthropi strain SM7.4, assembled by combining the Oxford Nanopore MinION flow cell and paired-end Illumina sequencing approaches.

A Genome Sequence Assembly of the Phototactic and Optogenetic Model Fungus Blastocladiella emersonii Reveals a Diversified Nucleotide-Cyclase Repertoire.

The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

Whole-genome sequencing of presumptive MDR-TB isolates from a tertiary healthcare setting in Mumbai

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB), proven to be a better alternative when compared with the combined sensitivity and specificity of all other modalities for diagnosis of tuberculosis (TB), aids epidemiological surveillance investigations by combining the current research with diagnostics.This study was conducted to identify and resolve operational challenges in performing WGS-based drug resistance testing (DRT) for MTB in a TB culture and drug susceptibility testing (DST) laboratory. Three critical, non-redundant steps for WGS-based DRT were tested: viz. DNA extraction, high-throughput paired-end next-generation sequencing (NGS), and genomic analysis pipeline for automated reporting of WGS-based DRT. DNA was extracted from 100 liquid culture isolates on a mycobacterial growth indicator tube (MGIT) using DNEASY Ultraclean Microbial Kit (Qiagen, USA) as per the manufacturer's instructions. Illumina paired-end sequencing was performed. All analysis steps were automated using custom python scripts, requiring no intervention. Variant calling was performed as per the World Health Organization (WHO) technical guide. The number of cultures resistant to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin was 89, 88, 35, 67, and 73, respectively. Resistance to amikacin, kanamycin, and capreomycin was found in 15, 17, and 15 cultures, respectively. Seventy cultures were resistant to fluoroquinolones, four were resistant to ethionamide, and 12 were resistant to linezolid. Six cultures were resistant to only one of the 18 drugs tested. Seventy-five cultures were resistant to more than three anti-TB drugs. One culture was resistant to 13 of the 18 anti-TB drugs tested for this study. The maximum number of variants were observed in the rpoB gene (n=93, 93%), wherein the Ser450Leu was the predominant mutation (n=68, 73%). Ser315Thr was the most common variant (n=86, 97%) that encoded resistance to isoniazid. The Lys43Arg variant encodes resistance to streptomycin and was the third most predominant variant (n=65, 89%). In addition to the high levels of resistance observed in the dataset, we also observed a high proportion of Beijing strains (n=63, 63%). Compared with results from routine diagnostics based on the 'Guidelines on Programmatic Management of Drug-Resistant TB (PMDT) in India', none of the samples had DST available for all 18 drugs. This represents a gap in PMDT guidelines. The WGS-DRT must be considered as the primary DST method after a sample is flagged rifampicin-resistant by cartridge-based nucleic acid amplification testing (CBNAAT).With several research studies currently underway globally to identify novel variants associated with drug resistance and classifiy their minimum inhibitory coefficients, WGS-DRT presents a scalable technology that updates analytical pipelines, relegating the need for changing microbiological protocols.

Whole genome sequencing of ASD 16 and ADT 43 to identify predominant grain size and starch associated alleles in rice.

The rice cultivars ASD16 and ADT43 are the most popular high-yielding Indica rice cultivars in southern India. Despite their popularity very little is known about their genetic basis due to lack of studies on the complete genome. In the current study, efforts were made to identify alleles and SNP markers that differentiate the two contrasting rice genotypes, ASD 16 and ADT 43 for grain shape and starch content. The complete genome of bold grain ASD16 and slender grain ADT43 were sequenced via Illumina's paired-end sequencing and the reads obtained were mapped to the Oryza sativa Indica Group cultivar 93-11 reference genome. The grain size of rice is controlled by Quantitative Trait Loci (QTL) that has a robust effect on grain yield and quality. To gain insight into genes that controlling grain size and starch content, an in-silico analysis was performed by taking into account of 72 grain elongation and starch biosynthesis genes. The identified alleles were further validated in the whole genome sequencing data of 32 bold grain and 25 slender grain varieties that were retrieved from the 3K rice genome project. An "A to G" polymorphism leading to SER 74 PRO was identified at the CDS position 220 of the An-1 gene, encoding bHLH domain-containing protein that regulates awn formation and increase in grain length. The non-synonymous substitutions such as A545C variant leading PHE 182 CYS in ADP Glucose Pyrophosphorylase large subunit IV (AGPL4) and C3094G variant leading to VAL 1032 LEU in Starch synthase IIIb (OsSSIIIb) were also identified in the starch biosynthesis genes. These identified allelic variants may contribute to the crop improvement programs in rice.

Complete-genome sequence and in vitro probiotic characteristics analysis of Bifidobacterium pseudolongum YY-26

The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential. Bifidobacterium pseudolongum YY-26 (CGMCC 24310) was isolated from faeces of mice treated with low-molecular-weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole-genome sequencing obtained using PacBio's single-molecular and Illumina's paired-end sequencing technology. A genome of 2.1Mb in length, with 1877 predicted protein-coding sequences was obtained. Carbohydrate-Activity enZyme analysis revealed that YY-26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY-26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short-chain fatty acid production was observed with single YY-26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY-26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging. B. pseudolongum YY-26 was identified with numerous probiotic-associated genes and its probiotic characteristics were verified in vitro. This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY-26.

A high-quality genome assembly of Lactarius hatsudake strain JH5.

Lactarius hatsudake is a species of Lactarius commonly found in pine forests, is edible with a delicious and nutritious fruiting body, and exhibits medicinal properties. It is an ideal natural multifunctional food with bioactive components including fungal polysaccharides, crude fiber, unsaturated fatty acids, nucleic acid derivatives, various amino acids, and vitamins. However, biological and genomic analyses of this mycorrhizal mushroom are sparse, thereby hindering large-scale cultivation. Previously, we isolated and screened L. hatsudake JH5 strains and have applied our garnered knowledge to the large-scale cultivation of mycorrhizal seedlings. In this study, we produced a high-quality genome assembly of L. hatsudake JH5 by combining Illumina paired-end and PacBio single molecule real-time sequencing, resulting in PacBio single molecule real-time reads of 7.67 Gb and Illumina Pair-End reads of 1,560 Mb. Based on the distribution of k-mer frequencies, the genome size of this strain was estimated to be 63.84 Mb (1.14% heterozygosity). Based on de novo genome assembly, the final genome size was determined to be 76.7 Mb, with scaffold N50 of 223.2 kb and N90 of 54.5 kb, and a GC content of 54.38%. BUSCO assessment showed that genome completeness was 89.0%. The N50 length of the JH5 genome was 43.6% longer than that of the previously published L. hatsudake MG20 genome. This high-quality L. hatsudake genome assembly will facilitate research on the functional genome, molecular breeding, yield enhancement, and sustainability of L. hatsudake cultivation.