Your Questions Answered
We’ve put together some frequently asked questions to give you more information about BASEwise Solutions and the services/products we offer. If your question is not listed here, then please contact us.
DNA Requirements
OmeSeq-qRRS
Bioinformatics / Analytics
Ordering Products & Services
DNA Requirements
For library preparation using OmeSeq-qRRS, we require a volume of 5 μl of DNA at a concentration of 20 ng/μl. However, we recommend sending at least 20 μl of each sample. Please ensure that sample concentrations do not exceed 50 ng/μL and are not less than 5 ng/μL. While samples with concentrations as low as 5 ng/μl, and even 1 ng/μl have been successfully sequenced, we do not guarantee success of library preparation outside the recommended range (5 – 50 ng/μL).
Currently, we do not offer a DNA extraction service. We will only accept DNA samples.
To save on time, we expect customers to determine DNA concentration and quality before shipping to us. If required, BASEwise Solutions can determine DNA concentration, perform normalization to 20 ng/μl, and ascertain restriction enzyme digestibility at a cost of $100 per plate. Ultimately, customers are responsible for sending DNA samples that meet quality specifications.
To determine DNA concentration, a florescence-based assay, such as PicoGreen, is preferred, as they typically yield more accurate and precise DNA estimations than absorbance-based methods. DNA quality can be ascertained by digesting a few random samples with any 4-bp restriction enzyme cutter, then running it alongside the undigested DNA control on a 1% agarose gel (or Bioanalyzer or TapeStation). A smear should be apparent in the digested DNA sample relative to the high molecular weight undigested control. At a minimum, we recommend you run at least 3 random samples per plate to assess DNA quality.
If library preparation fails, we will troubleshoot the source of the problem. If DNA meets all quality specifications, BASEwise Solutions will repeat the library preparation. If library preparation fails again and/or troubleshooting does not resolve the problem, we will not charge for the service.
Samples should be shipped in a 96-well plate format, as the assay is based on blocks of 96 samples. Should you have fewer than 96 samples, one option is to duplicate some samples to fill up remaining wells, although this is done at the expense of the customer. Ensure that plates are either semi-skirted or fully skirted, and properly sealed to avoid spillage during shipping. Please see our Sample Submission Guidelines for more detailed information.
OmeSeq-qRRS
At a minimum, we require a contig-level genome assembly for variant calling, even if assembly is highly fragmented. A reference genome for a different species within the same genus (maybe even within the same family) will work well as a reference genome for variant calling. In the absence of any reference genome, BASEwise Solutions can provide services that will include shotgun whole genome sequencing and contig level assembly for approximately $1,000 (depending on genome size and sequencing requirement).
Yes, OmeSeq-qRRS was initially developed for dosage-calling in polyploids. Depending on the genome size and ploidy level, you may need to sequence more deeply than in a diploid. This is because the read depth thresholds are typically higher in polyploids.
Any Illumina sequencing platform works for and is available for OmeSeq-qRRS. Typically, we recommend sequencing on NovaSeq 6000 SP or S4 flow cells.
Bioinformatics / Analytics
Demultiplexing and quality filtering of sequence reads are included in the library preparation + sequencing services we provide. Additional bioinformatic analyses, including variant calling and filtering; quantitative metagenome profiling down to strain-level; and whole genome assembly are also available.
You will receive demultiplexed FASTQ files and summary QC plots. If variant calling/filtering and/or metagenome profiling is required, appropriate output files and visualizations will be provided. For variant calling/filtering, variants will be provided in multiple formats i.e. VCF; Hapmap; and genotype files with dosage codes such as (0, 1, and 2 for diploids; 0,1, 2, 3, 4 for tetraploid; etc.); and genotype files with binary code (0/0, 0/1, and 1/1).
Yes. Both variant calling (SNPs) and metagenome profiling can conveniently be performed on the same data derived from OmeSeq-qRRS. Separate bioinformatic analyses for variant calling and metagenome profiling is all that is required to extract the relevant information.
We store your data for 3 months.
Ordering Products & Services
Library preparation of up to 12 x 96-well plates (1152 samples) takes 2-3 days. Sequencing takes 2-3 weeks. Turnaround time for bioinformatic analysis depends on sample size and sequencing coverage. Demultiplexing and read quality filtering takes 12 hours for 400 million reads and 3 days for 2.5 billion reads. Typically, variant calling/filtering of 384 samples takes about one week. Metagenome analysis depends on sample size, diversity of metagenomic community, size of database being used for taxonomic identification. Typically for 384 samples: (i) takes a few hours against the 16S/ITS databases, (ii) takes about 1-2 weeks against the NCBI nt + RefSeq database.
Yes. Detailed shipping information for international customers is available in our Sample Submission Guidelines.