Customer-friendly solutions

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).