Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Complete Genome Sequence of the Soil-Isolated Psychrobacillus sp. Strain AK 1817, Capable of Biotransforming the Ergostane Triterpenoid Antcin K

The soil bacterium Psychrobacillus sp. strain AK 1817 was isolated from a tropical soil sample collected in Taiwan. Strain AK 1817 biotransforms the ergostane triterpenoid antcin K from the fungus Antrodia cinnamomea . The genome was sequenced using the PacBio RS II platform and consists of one chromosome of 4,096,020 bp, comprising 3,907 protein-coding genes, 75 tRNAs, 30 rRNAs, 5 noncoding RNAs (ncRNAs), and 100 pseudogenes.

Complete Genome Sequence of Erwinia amylovora Strain TS3128, a Korean Strain Isolated in an Asian Pear Orchard in 2015

Erwinia amylovora causes fire blight, a devastating disease of apples and pears. Here, we report the complete genome sequence and annotation of E. amylovora strain TS3128, which was isolated from Anseong, South Korea, where fire blight first occurred in 2015, using the PacBio RS II system.

Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms

Studying isoform expression at the microscopic level has always been a challenging task. A classical example is kidney, where glomerular and tubulo-insterstitial compartments carry out drastically different physiological functions and thus presumably their isoform expression also differs. We aim at developing an experimental and computational pipeline for identifying isoforms at microscopic structure-level. We microdissed glomerular and tubulo-interstitial compartments from healthy human kidney tissues from two cohorts. The two compartments were separately sequenced with the PacBio RS II platform. These transcripts were then validated using transcripts of the same samples by the traditional Illumina RNA-Seq protocol, distinct Illumina RNA-Seq short reads from European Renal cDNA Bank (ERCB) samples, and annotated GENCODE transcript list, thus identifying novel transcripts. We identified 14,739 and 14,259 annotated transcripts, and 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Of note, relying solely on either short or long reads would have resulted in many erroneous identifications. We identified distinct pathways involved in glomerular and tubulointerstitial compartments at the isoform level.We demonstrated the possibility of micro-dissecting a tissue, incorporating both long- and short-read sequencing to identify isoforms for each compartment.

Full-Length Transcriptome Sequencing Provides Insights into Flavonoid Biosynthesis in Fritillaria hupehensis

One of the most commonly utilized medicinal plants in China is Fritillaria hupehensis (Hsiao et K.C. Hsia). However, due to a lack of genomic resources, little is known about the biosynthesis of relevant compounds, particularly the flavonoid biosynthesis pathway. A PacBio RS II sequencing generated a total of 342,044 reads from the bulb, leaf, root, and stem, of which 316,438 were full-length (FL) non-redundant reads with an average length of 1365 bp and a N50 of 1888 bp. There were also 38,607 long non-coding RNAs and 7914 simple sequence repeats detected. To improve our understanding of processes implicated in regulating secondary metabolite biosynthesis in F. hupehensis tissues, we evaluated potential metabolic pathways. Overall, this study provides a repertoire of FL transcripts in F. hupehensis for the first time, and it will be a valuable resource for marker-assisted breeding and research into bioactive compounds for medicinal and pharmacological applications.

Complete Genome Analysis of Flavobacterium psychrophilum Strain FPRT1, Isolated from Diseased Rainbow Trout (Oncorhynchus mykiss) in South Korea

ABSTRACT Here, we report the complete genome sequence of Flavobacterium psychrophilum FPRT1, isolated from the spleen and kidney of diseased rainbow trout (Oncorhynchus mykiss). Whole-genome sequencing was performed using the PacBio RS II platform, which yielded a circular chromosome of 2,795,347 bp harboring 2,895 protein-coding genes.

Complete Genome Sequence of Escherichia coli Strain FEX669, a ColV Plasmid-Containing Isolate from Retail Chicken Meat

ABSTRACT Escherichia coli strain FEX669 was isolated from retail ground chicken and shown to contain the extraintestinal pathogenic E. coli (ExPEC) virulence genes sfaD, focC, and iutA. Because this presumptive ExPEC strain was isolated from a retail food item and it was a weak biofilm former, it was characterized using whole-genome sequencing using the PacBio RS II platform. Genomic analysis showed that the FEX669 chromosome is 4,973,943 bp long, with a GC content of 50.47%, and is accompanied by a ColV plasmid that is 237,102 bp long, with a GC content of 50.49%.

Improved High-Throughput Sequencing of the Human Oral Microbiome: From Illumina to PacBio

Background. A comprehensive understanding of the commensal microflora and its relation to health is essential for preventing and combating diseases. The aim of this study was to examine the structure of the oral microbiome by using different sequencing technologies. Material and Methods. Five preschool children with no symptoms of oral and systemic diseases were recruited. Samples of saliva were collected. A 468 bp insert size library was constructed on the MiSeq platform and then subjected to 300 bp paired-end sequencing. Libraries with longer insert sizes, including a full-length 16S rDNA gene, were sequenced on the PacBio RS II platform. Results. A total of 122.6 Mb of raw data, including 244,967 high-quality sequences, were generated by the MiSeq platform, while 134.6 Mb of raw data, including 70,030 high-quality reads, were generated by the PacBio RS II platform. Clustering of the unique sequences into OTUs at 3% dissimilarity resulted in an average of 225 OTUs on the MiSeq platform; however, the number of OTUs generated on the PacBio RS II platform was 449, far greater than the number of OTUs generated on the MiSeq platform. A total of 437 species belonging to 10 phyla and 60 genera were detected by the PacBio RS II platform, while 163 species belonging to 12 phyla and 72 genera were detected by the MiSeq platform. Conclusions. The oral microflora of healthy Chinese children were analyzed. Compared with traditional 16S rRNA sequencing technology, the PacBio system, despite providing a lower amount of clean data, surpassed the resolution of the MiSeq platform by improving the read length and annotating the nucleotide sequences at the species or strain level. This trial is registered with NCT02341352.

Analysis of HLA-G long-read genomic sequences in mother–offspring pairs with preeclampsia

Preeclampsia is a pregnancy-induced disorder that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. HLA-G is thought to play important roles in maternal–fetal immune tolerance, and the associations between HLA-G gene polymorphisms and the onset of pregnancy-related diseases have been explored extensively. Because contiguous genomic sequencing is difficult, the association between the HLA-G genotype and preeclampsia onset is controversial. In this study, genomic sequences of the HLA-G region (5.2 kb) from 31 pairs of mother–offspring genomic DNA samples (18 pairs from normal pregnancies/births and 13 from preeclampsia births) were obtained by single-molecule real-time sequencing using the PacBio RS II platform. The HLA-G alleles identified in our cohort matched seven known HLA-G alleles, but we also identified two new HLA-G alleles at the fourth-field resolution and compared them with nucleotide sequences from a public database that consisted of coding sequences that cover the 3.1-kb HLA-G gene span. Intriguingly, a potential association between preeclampsia onset and the poly T stretch within the downstream region of the HLA-G*01:01:01:01 allele was found. Our study suggests that long-read sequencing of HLA-G will provide clues for characterizing HLA-G variants that are involved in the pathophysiology of preeclampsia.

Whole-Genome Sequencing of Lactobacillus helveticus D75 and D76 Confirms Safety and Probiotic Potential

Whole-genome DNA sequencing of Lactobacillus D75 and D76 strains (Vitaflor, Russia) was determined using the PacBio RS II platform, which was followed by de novo assembly with SMRT Portal 2.3.0. The average nucleotide identity (ANI) test showed that both strains belong to the Lactobacillus helveticus, but not to the L. acidophilus, as previously assumed. In addition, 31 exopolysaccharide (EPS) production genes (nine of which form a single genetic cluster), 13 adhesion genes, 38 milk protein and 11 milk sugar utilization genes, 13 genes for and against specific antagonistic activity, eight antibiotic resistance genes, and also three CRISPR blocks and eight Cas I-B system genes were identified in the genomes of both strains. The expression of bacteriocin helveticin J genes was confirmed. In fact, the presence of identified genes suggests that L. helveticus D75 and D76 are able to form biofilms on the outer mucin layer, inhibit the growth of pathogens and pathobionts, utilize milk substrates with the formation of digestible milk sugars and bioactive peptides, resist bacteriophages, show some genome-determined resistance to antibiotics, and stimulate the host’s immune system. Pathogenicity genes have not been identified. The study results confirm the safety and high probiotic potential of the strains.