Whole Genome

Plasmidsaurus Whole Genome Sequencing is performed using the newest long-read sequencing technology from Oxford Nanopore Technologies (ONT).

We use a transposome complex to construct an amplification-free long-read sequencing library, including minimal fragmentation of the input genomic DNA in a sequence-independent manner, and return you the annotated genome assembly. For more details on our workflows and bioinformatics pipelines, please see technical documentation for Whole Genome Sequencing. 

We require a minimum raw read Qscore of 10 (90% accuracy) during sequencing, although most raw reads are above Q20 (99% accuracy). We use ONT’s super-accurate basecalling model.

During assembly, we filter out the bottom 5% of reads based on quality (see technical documentation for more details). If sufficient coverage to meet our target is obtained, we typically see assembled contigs with ~Q40 (99.99%) accuracy.

We can obtain even higher accuracy in the known error-prone homopolymers and methylated motifs with our Hybrid sequencing option that polishes with Illumina data.

Briefly, our process follows a number of steps:

• Multiple rounds of quality score filtering
• Assembling the remaining best reads with assembly algorithms
• Polishing the assembly using parameters selected for high quality nanopore reads
• For hybrid service, the assembly is then polished again using short reads
• Assessment of contig quality, genome completeness, and contamination
• Genome annotation with multiple tools

Check out our technical documentation for more details.

The typical turnaround time in business days is listed below for each service. This is the average time it takes to return data to customers who are submitting orders from US, UK and EU. Turnaround times for customers in APAC may be slightly longer due to the time required in transit to our lab in Singapore.

Low sample quality can also negatively impact turnaround time and the quality of your results. Please read sample prep instructions carefully to make sure your samples meet input requirements. Due to variability in shipping logistics and sample quality that is outside our control we cannot guarantee turnaround times or sample success rates.

Our mission is to accelerate science, so we work to get answers back as fast as possible. Rarely, your results might be delayed longer than due to:

• A shipping issue that causes your samples to be delayed in transit from your location to the local lab.
• Your samples may need to be internally reshipped from your local lab to a different lab location for processing the particular service you’ve requested.
• Your samples have low concentration, quality, or purity that requires a longer sequencing run to increase chances of success. Please refer to sample prep instructions in order to ensure that your samples meet the requirements for sequencing. 

This service is intended for sequencing of clonal isolates. It is hard to predict the assembly outcome if you send a mixture, but it most likely won't work particularly well. If you send mixed samples at your own risk they will not be eligible for free rerun if your desired outcome is not produced.

For bacteria, we have a Microbiome 16S service for community profiling. We are able to perform metagenomic shotgun sequencing via our Custom Sequencing Service.

Our bacterial, yeast, and eukaryotic services offer similar library preparation and sequencing quality but specialized genome extraction services, sequencing depth, and bioinformatics pipelines optimized for bacteria, yeast, and eukaryotes (including filamentous fungi and multicellular animals). 

We recommend choosing the option that matches the organism you are sequencing. If you need more read depth for yeast sequencing, you can submit yeast samples to the Eukaryotic genome services that offer higher data targets. Deeper sequencing is also available for multicellular genomes via our Custom Service. 

The Hybrid ONT + Illumina option for Bacteria and Yeast Genome Sequencing polishes your long-read ONT assembly with Illumina short reads (paired-end 2x150bp) using the Polypolish v0.6 tool to improve the consensus quality at those tricky homopolymer runs and methylation motifs. 

This Hybrid service is ideal when:

• You anticipate an abundance of homopolymers and/or methylated motifs in the genome
• You are planning to use the genome assembly result as a reference for SNP variant calling

The Hybrid service is not generally necessary if your primary goal is to obtain a high quality, high coverage, highly contiguous genome assembly, as contiguity is entirely determined by the quality and quantity of the ONT long read data. Adding in short Illumina reads with the Hybrid service has no impact on the number, size, or topology of assembled chromosomes/elements, and instead only improves nucleotide-level basecalling accuracy in homopolymers and methylated motifs via polishing. For this reason, we do not repeat the genome assembly or contig identification workflows after Illumina polishing.
 
Please note: Hybrid ordering options are not currently available for the Eukaryotic Genome, but you can request to add Illumina data to your ONT eukaryotic data through the Custom Sequencing service.

Yes. The assembled contigs will indicate the presence, orientation, and copy number of all genes, making it straightforward for you to confirm genetic modifications.

While we do not guarantee that you will receive a minimum amount of sequencing data in your results, our internal data targets for bacterial genome sequencing are:

• Standard: 210 Mb of ONT raw data
• Big: 360 Mb of ONT raw data
• Hybrid: 210 Mb of ONT raw data + 100 Mb of Illumina raw data
• Big Hybrid: 360 Mb of ONT raw data + 170 Mb of Illumina raw data

We sequence all molecules in the received sample without primers, so if your extracted bacterial DNA also contains plasmid DNA, then yes you will probably receive some plasmid reads. Most of the DNA fragments < 3kb are omitted during data processing, but otherwise we do not select against or omit plasmid-sized reads during sequencing or assembly.

The number of raw reads produced by each type of DNA will vary based on their relative abundance and quality. As for assembly outcomes, we do usually see that plasmid contigs are produced along with the gDNA chromosome contig(s). However, since this bacterial genome sequencing service is optimized for assembly of the chromosomal genome (not for plasmids), we cannot guarantee that the raw plasmid reads will always yield an assembled plasmid contig. If you do need assemblies for the plasmids, you may need to isolate reads that align to your expected plasmids and assemble them yourself with a different pipeline.

Ultimately, when submitting mixtures, which types of DNA in your sample end up producing an assembled contig will vary depending on overall sample quality, coverage, and relative abundance/degradation of each type.
 

Yes, our assembly workflow does typically produce a closed genome with a full-length chromosome contig. Samples that are low quality or produce low coverage, or that contain extrachromosomal DNA, inserted DNA elements, mixed genotypes, and/or ambiguous genomic elements (such as repetitive regions) may inhibit production of a closed genome.

We recommend ordering big for unknown species, as this increases chances of getting sufficient coverage if the genome ends up being on the larger end.

Yes! You can use the genome alignment tool to compare any two bacterial genomes. You’ll just need to first download one of the samples and then upload it as a reference.

Yes! Because this tool sits in the results page, at least one of the two sequences to compare must be a Plasmidsaurus sample.

If you would like to align any two sequences, please use our new pairwise alignment tool.

While we do not guarantee that you will receive a minimum amount of sequencing data in your results, our internal data targets for yeast genome sequencing are:

• Standard: 600 Mb of ONT raw data
• Hybrid: 600 Mb of ONT raw data + 290 Mb of Illumina raw data

We sequence all molecules in the received sample without primers, so if the post extraction DNA also contains plasmids or YACs, then this will also be sequenced. Please note that in order to improve assemblies, most of the sequenced DNA fragments < 3kb are omitted during data processing, but otherwise we do not select against or omit plasmid-sized reads during sequencing or assembly.

The number of raw reads produced by each type of DNA will vary based on their relative abundance and quality. As for assembly outcomes, we frequently see plasmid or YAC contigs produced along with the gDNA chromosome contigs during assembly. However, since this service is optimized for assembly of the chromosomal genome, we cannot guarantee that these raw plasmid reads will always yield an assembled plasmid or YAC contig. 

If you do need assemblies for the plasmids or YACs, it is possible isolate raw reads that align to your expected references and assemble them. Ultimately, when submitting mixtures, which types of DNA in your sample end up producing an assembled contig will vary depending on overall sample quality, coverage, and relative abundance of each type.

If you would like to discuss options for optimising the number of non-chromosomal reads via our Custom service, please submit a project inquiry via our Custom Sequencing page. 

The Eukaryotic Genome Sequencing service offers four different service levels,  which allow you to select the target amount of output per sample, measured in Gb of fastq data. We don't specify output in number of reads because that varies based on the specific read length distribution in question. 

The Service Level recommendations below are based on a target of approximately 30x genome coverage, which is typically sufficient to generate a high-quality de novo genome assembly with annotations. Depending on your application, you can submit your sample to a different service level than is recommended in order to obtain a different amount of coverage. For example:

• If you plan to use the raw reads to perform variant analysis against a reference genome, you might need less genome coverage, e.g. 10-20x. Please note that while the assembly is more likely to fail at lower coverage levels, you will still be able to use the raw reads to perform your analysis
• If you plan to use the raw reads to generate a haplotype-phased de novo genome assembly, in some cases you might need more genome coverage, e.g. 60-100x

“Fail” samples are defined by achieving neither the target amount of raw data, or any high coverage, high quality contigs. We still provide the raw data and the assembly report for failed samples, and you may also still receive some of the other file types.

Although we do not provide definitive reasons on why each specific sample failed (or had low coverage or otherwise poor results), by far the most common reasons are:

• Your samples are not shipped at the required DNA concentration of 50 ng/uL.
  • The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent fluorometric assay.
• The gDNA in your samples is degraded or fragmented.
  • At least 50% of the DNA should be above 15kb in length, and samples should be handled with utmost care:
    • Pipetting with wide-bore tips
    • Minimal freeze/thaw cycles
    • No vortexing
    • No extreme temperature/pH
    • No intercalating dyes
    • No UV radiation
    • Not over-dried
• Your samples contain inhibitors, such as:
  • RNA
  • Denaturants (guanidinium salts, phenol, etc.)
  • Detergents (SDS, Triton-X100, etc.)
  • Residual contaminants from the organism/tissue (heme, humic acid, polyphenols, polysaccharides, lipids, etc.)
  • Insoluble, colored, or cloudy material
  • Other inhibitors (EDTA, etc.)
• The DNA you sent is not from a single isolate.
  • This service is intended for a clonal population (single species). If your sample contains a mixture of different species, it may fail to produce an assembly.
• For the extraction option: You did not ship us the required number of cells
  • Please perform a cell count while preparing your preserved cells to confirm that you are sending the number of cells we require.

To increase chances of successful sequencing on the first attempt, please adhere closely to our sample prep guidelines and cell pellet guidelines.

You are welcome to submit a rerun request for any failed sample through your Order Info page or via the support@plasmidsaurus.com email address. We will evaluate whether you sample quality and quantity permits rerunning the sample (and we may also ask you to provide a reference sequence).

Sample quality checks may require any of the following: 

• Quantify concentration and/ or purity
• Sample clean-up using the Bead Clean Up Protocol ($5 per sample)
• Normalizing concentration

If you have questions about your results, please get in touch at support@plasmidsaurus.com