Single Molecule, Real-Time (SMRT) Sequencing uses the natural process of DNA replication to sequence long fragments of native DNA in order to produce highly accurate long reads, or HiFi reads. As such, starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in longer libraries and better performance during sequencing. This technical note is intended to give recommendations, tips and tricks for the extraction of DNA, as well as assessing and preserving the quality and size of your DNA sample to be used for HiFi sequencing.
The Agilent 5200, 5300, and 5400 Fragment Analyzer instruments are fast, high-resolution benchtop capillary electrophoresis (CE) platforms that utilize proprietary markers to accurately size fragments ranging from 10 to 50 kb. This platform allows important DNA quality checkpoints to be completed in one hour for de novo large-genome sequencing projects and other PacBio applications leveraging multi-kilobase read lengths. The instrument can be used in place of time-consuming QC steps involving pulsed field gel electrophoresis (PFGE), saving time by avoiding multiple overnight gel runs when preparing large-insert SMRTbell libraries. Alternative DNA-sizing instruments cannot accurately resolve large DNA fragments in this range.
The Agilent Femto Pulse system automated pulsed-field CE instrument is a fast, high-resolution benchtop capillary electrophoresis (CE) platform that utilizes pulsed-field electrophoresis to separate high molecular weight DNA fragments. This platform allows important DNA quality checkpoints to be completed in less than 1.5 hours with minimal sample input for de novo large genome sequencing projects and other PacBio applications leveraging multi-kilobase read lengths. The instrument can be used in place of gel-based pulsed-field electrophoresis (PFGE) systems to fully support generation of large-insert SMRTbell libraries with accurate sizing to 165 kb. Alternative DNA sizing instruments cannot accurately resolve large DNA fragments…
PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated…
Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on…
Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in…
Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the…
An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with…
In this webinar you will hear how several researchers have overcome the challenges of sequencing organisms with small body size using the new low and ultra-low DNA input methods from PacBio. Learn about the advantages of using highly accurate long reads (HiFi reads) to sequence and de novo assemble genomes of single individuals.
Chlorobenzenes are ubiquitously distributed, highly persistent, and toxic environmental contaminants. Pandoraea pnomenusa MCB032 was isolated as a new dominant chlorobenzene-utilizing strain from a functionally stable bioreactor during the treatment of chlorobenzenes when strain Burkholderia sp. JS150 disappeared. In study, we report the complete genome sequence of strain MCB032 which consists of a circular chromosome and three plasmids, which are?~?6 Mb in length with 5450 open reading frames-12 encoding rRNAs and 77 encoding tRNAs. We further identified 17 putative genes encoding the enzymes involved in the methyl-accepting chemotaxis proteins in sensing chemical gradients during chemotaxis. The annotated complete genome sequence of this…
Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and…
Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled…
Marinobacter sp. strain LQ44, an alkaliphile and moderate halophile from a deep-sea hydrothermal vent on the East Pacific Rise, is a novel phenol-degrading bacterium that is capable of utilizing phenol as sole carbon and energy sources. Here, we present the complete genome sequence of strain LQ44, which consists of 4,435,564?bp with a circular chromosome, 4164 protein-coding genes, 3 rRNA operons and 50 tRNAs. Genome analysis revealed that strain LQ44 may degrade phenol via meta-cleavage pathway. The LQ44 genome contains multiple genes involved in pH adaptation and osmotic adjustment. Genes related to hydrocarbon degradation, aerobic denitrification and potential industrial important enzymes…
Paracoccus sp. Arc7-R13, a silver nanoparticles (AgNPs) synthesizing bacterium, was isolated from Arctic Ocean sediment. Here we describe the complete genome of Paracoccus sp. Arc7-R13. The complete genome contains 4,040,012?bp with 66.66?mol%?G?+?C content, including one circular chromosome of 3,231,929?bp (67.45?mol%?G?+?C content), and eight plasmids with length ranging from 24,536?bp to 199,685?bp. The genome contains 3835 protein-coding genes (CDSs), 49 tRNA genes, as well as 3 rRNA operons as 16S-23S-5S rRNA. Based on the gene annotation and Swiss-Prot analysis, a total of 15 genes belonging to 11 kinds, including silver exporting P-type ATPase (SilP), alkaline phosphatase, nitroreductase, thioredoxin reductase, NADPH dehydrogenase…
Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It…