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Metagenomics / Genomics

Whole-genome sequencing, de novo assembly and bioinformatic analysis of either clonal cultures (genomics) or microbial communities (metagenomics) not only provides a detailed blueprint of the metabolic potential (genes and metabolic pathways) but is also often a prerequisite for studying gene expression patterns (transcriptomics or proteomics) and essential for high resolution strain typing and comparative genomics.

The team behind DNASense has extensive experience within the fields of genomics and metagenomics and our active involvement in state-of-the-art methods and sequencing platforms ensures that customers obtain valuable insight from our tailored bioinformatic analyses.

We offer access to both short-read Illumina (MiSeq, HiSeq and NovaSeq) and long-read (Oxford Nanopore and PacBio) DNA sequencing platforms, allowing us to tailor sequencing and bioinformatic workflows according to your specific requirements.

For reference grade assemblies and metagenomic binning we currently recommend a hybrid sequencing approach involving Illumina short-read and Oxford Nanopore long-read. This greatly assists the retrieval of high-quality, contiguous metagenome-assembled genomes (MAGs) and eliminates GC-, amplification-, and loading-biases (accurate abundance estimates).

Pure culture genome price example: Standard package price example for pure culture sequencing and analysis of typical bacterial-sized genomes (4-5 Mbp). We recommend 100x coverage for de novo assembly and 50x Illumina for polishing of Oxford Nanopore assemblies (hybrid reference grade assemblies).

Technology Assembly strategy Assembly contiguity Consensus accuracy Turnaround time* List price/sample**
Illumina Short-read Typically fragmented > 99.99 % 5 weeks 400 / 300 EUR
Oxford Nanopore Long-read Highly contiguous > 99.9 % 2 weeks 300 / 250 EUR
Illumina + Oxford Nanopore Hybrid Highly contiguous > 99.99 % 5 weeks 600 / 450 EUR

*Estimated turnaround time, **The two listed prices correspond to a minimum of 12 and 24 samples, respectively.

Metagenome price example: Standard package price example for sequencing and analysis of one metagenome with either 12 or 24 sample dimensions. The price assumes that 100x coverage of a bacterial-sized genome (4-5 Mbp) present at 3 % abundance is needed (15 Gbp Illumina or Oxford Nanopore data) along with 50x Illumina coverage pr. sample dimension to assist genome binning (genome binning is an add-on feature).

Technology Assembly strategy Assembly contiguity Consensus accuracy Turnaround time* List price/sample**
Illumina Short-read Typically fragmented > 99.99 % 5 weeks 500 / 400 EUR
Illumina + Oxford Nanopore Hybrid Highly contiguous > 99.99 % 5 weeks 600 / 450 EUR
Oxford Nanopore Long-read Highly contiguous > 99.9 % 3 weeks enquire

*Estimated turnaround time, **The two listed prices correspond to a minimum of 12 and 24 samples, respectively.

Our standard package includes: Optional pre- and post-project meeting with a DNASense specialist, DNA extraction, library preparation, sequencing, pre- and post-sequencing quality control, de novo assembly, taxonomic profiling, gene annotation, online-access to raw data and result files and a detailed project report.

Add-on services (non-exhaustive list): Tailored DNA extraction and purification, genome binning, SNP-calling, Functional annotation (KO, GO and KEGG), functional enrichment analysis, manual curation of metabolic pathways, gene mining, core-genome SNP analysis, multi-locus sequencing typing (MLST), custom annotation, epigenetic analysis, data submission.

Pure culture genome price example: Standard package price example for pure culture sequencing and analysis of typical bacterial-sized genomes (4-5 Mbp). We recommend 100x coverage for de novo assembly and 30x Illumina for polishing of Oxford Nanopore assemblies (hybrid reference grade assemblies).

Technology Assembly strategy Assembly contiguity Consensus accuracy Turnaround time* List price/sample**
Illumina Short-read Typically fragmented > 99.99 % 5 weeks 3000 / 2250 DKK
Oxford Nanopore Long-read Highly contiguous > 99.9 % 2 weeks 2250 / 1900 DKK
Illumina + Oxford Nanopore Hybrid Highly contiguous > 99.99 % 5 weeks 4500 / 3350 DKK

*Estimated turnaround time, **The two listed prices correspond to a minimum of 12 and 24 samples, respectively.

Metagenome price example: Standard package price example for sequencing and analysis of one metagenome with either 12 or 24 sample dimensions. The price assumes that 100x coverage of a bacterial-sized genome (4-5 Mbp) present at 3 % abundance is needed (15 Gbp Illumina or Oxford Nanopore data) along with 30x Illumina coverage pr. sample dimension to assist genome binning (genome binning is an add-on feature).

Technology Assembly strategy Assembly contiguity Consensus accuracy Turnaround time* List price/sample**
Illumina Short-read Typically fragmented > 99.99 % 5 weeks 3750 / 2250 DKK
Illumina + Oxford Nanopore Hybrid Highly contiguous > 99.99 % 5 weeks 4450 / 3350 DKK
Oxford Nanopore Long-read Highly contiguous > 99.9 % 3 weeks enquire

*Estimated turnaround time, **The two listed prices correspond to a minimum of 12 and 24 samples, respectively.

Our standard package includes: Optional pre- and post-project meeting with a DNASense specialist, DNA extraction, library preparation, sequencing, pre- and post-sequencing quality control, de novo assembly, taxonomic profiling, gene annotation, online-access to raw data and result files and a detailed project report.

Add-on services (non-exhaustive list): Tailored DNA extraction and purification, genome binning, SNP-calling, Functional annotation (KO, GO and KEGG), functional enrichment analysis, manual curation of metabolic pathways, gene mining, core-genome SNP analysis, multi-locus sequencing typing (MLST), custom annotation, epigenetic analysis, data submission.

FAQ

  • For clonal (pure) culture genomics, we recommend targeting a sequencing depth corresponding to 100x assembly coverage (500 Mbp for a 5 mbp genome). Short-read (2×150 bp) DNA sequencing will provide a high-quality but fragmented assembly (several contigs). If highly contiguous or closed reference-grade genomes are desired, we recommend combining short-read Illumina data (50x) with long-read Nanopore sequencing data (50-100x).

  • Depending on the question asked, we recommend a sequencing depth of 100x. If you are interested in studying a microorganism (5 Mbp genome) present at a 1 % abundance, you would then need 50 Gbp data ([100/abundance] * [genome size] * [depth]).

  • While it is possible and the less-biased approach, you would need a relative high sequencing depth. Instead, consider using an amplicon-based approach. It is more sensitive, and you would need less data.

  • If your metagenome is relatively enriched (2-10 microorganisms) we can usually bin genomes from a combination of GC content, coverage and using more advanced tSNE plots. For more diverse metagenomes, we need additional sample dimensions to separate the genome bins. This could reflect different sample timepoints or samples extracted using different methods.

  • To be statistical meaningful, we recommend a 50-fold read depth.

  • We use the Qubit high-sensitivity dsDNA assay, which is specific for double-stranded DNA. Standard spectrophotometers based on absorbance at 260 nm is generally not suitable for estimating the concentration of DNA or RNA.

  • For Illumina paired-end sequencing (2×150 bps) we recommend concentrations above 2 ng/µL (20 µL) but less might possible. For long-read sequencing, it depends heavily on the details. Typically, 100-2000 ng.

  • For low input DNA samples, we always recommend including a DNA extraction negative to assess the impact of potential kit contaminants.

  • It varies. For thin water-samples, we can filter large sample volumes to concentrate the biomass. For high-load biomass samples, Eppendorf-tube pellets often suffices. Whenever possible, we generally prefer to have or make a backup in case of unforeseen obstacles or challenges.

  • For relatively complete prokaryotic genomes with little or no contamination, we use the Genome Taxonomy Database (GTDB), which potentially provides species-level resolution. Our standard service also includes rDNA extraction and classification against the Silva SSU database (genus level for both prokaryotes and eukaryotes). Custom databases can be included (add-on service).

  • Our standard library preparation protocol involves sequencing native DNA. There is no amplification or tagmentation involved.

  • Absolutely. We recommend Nanopore as there is minimal compositional biases associated with this sequencing platform.

  • Nanopore sequencing yield depends on many factors pertaining to the nature of the (native) DNA being sequenced. Therefore, we cannot offer any guarantee, but we regularly generate 20-30 Gbp on a single MinION run.

  • Unless agreed otherwise, we follow a thorough community-adopted bead-beating protocol to reduce the (potentially large) DNA extraction bias. This often results in extended DNA shearing but is still compatible with the generation of very contiguous prokaryotic assemblies from long-read sequencing platforms.

  • It depends on the aim of your analysis. If you wish to produce closed genomes, your DNA read length distribution should be compatible with spanning the longest repeat element in your target genome. For bacteria, this is often the rRNA operon, i.e., reads should be able to span a length of 5000-7000 bp.

  • The raw read accuracy of Nanopore sequencing is lower that Illumina but has comparable consensus accuracy. Nanopore still has a few systematic errors pertaining to regions involving homopolymers. These remaining errors can be handled by polishing with a small amount of Illumina data (30x).

Rasmus Dam Wollenberg, Ph.D.
Chief Technology Officer

For more details please check out our metagenomics / genomics product sheet

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