Customer documentation portal

For duet products, each kit consists of 2 boxes:

• 1 room temperature box to be stored at room temperature and
• 1 frozen box which is shipped on dry ice and to be stored at -20C.

For duet mosaic products:

• each kit includes a frozen box shipped on dry ice, which should be stored at -20°C upon receipt.
• For kits ordered with magnetic beads, an additional room temperature box is supplied and should be stored at room temperature.

There may be additional storage instructions for specific reagents, which we will highlight as necessary.

There is one additional box for the UDIs shipped on dry ice and to be stored at -20C but this is a separate product.

US shipments will dispatch from Tennessee, USA on Monday - Thursday for next day delivery. EU shipments will dispatch from the Netherlands with expected lead times of 24-48 hours transit times across Europe.

Shipments to locations such as Australia and Hong Kong can be anywhere between 1-3 weeks (please note that the kit is in transit for 3-5 days, the additional time is needed for customs paperwork and arranging the shipment). 

Kits are shipped with a minimum of 3 months shelf life remaining.

As few as possible, we recommend no more than 3.

No, improper storage temperature has a detrimental effect on buffer and enzyme activity. Get in touch with us to discuss the issue and arrange replacements.

Please get in touch with us immediately if any tubes have insufficient volumes so we can arrange a solution. If you have noticed this while carrying out the prep, depending on the reagent, you may still be able to store the partly processed DNA sample until the replacement reagents have reached you. Please get in touch with our support team to advise.

The required reagents, consumables and equipment are listed in the Kit Information section of the relevant User Guide. Please refer to the latest version of the User Guide for your product for the most up-to-date information.

Our assay is optimised for mechanical shearing, we have not validated our assays with enzymatic fragmentation. 

We recommend using Covaris M220 when possible as we have found it to be the most reliable and we have optimised our assay with it. We have had customers use other fragmentation instruments, such as a Bioruptor. We recommend to always check fragment size after shearing using a Bioanalyser, TapeStation, or similar.

Yes, SPRIselect beads can be used as substitute. Please follow the bead ratios and clean up steps described in our user guides.

duet can be used in conjunction with the Twist Alliance Pan-Cancer panel (1.5Mb) and the Human Methylome panel (123Mb). We have supplementary user guides available on our customer documentation portal, to be used alongside the main duet evoC/+modC user guides.

In addition to the duet reagents, you will need to purchase the relevant Twist panel and the following Twist reagents, as specified in the Twist Targeted Methylation Sequencing Protocol:

• Twist Fast Hybridization Reagents
• Twist Fast Wash Buffers
• Equinox Library Amp Mix
• Twist Universal Blockers
• Twist Binding and Purification Beads
• Twist Methylation Enhancer

• Recommended input range for gDNA (duet products only) is 10-80 ng of double-stranded DNA quantified post-sonication.
• Recommended input range for cfDNA is 5-30 ng. Any input beyond that range is at the customer's risk/discretion.

This duet kit is compatible with gDNA and cfDNA. The duet mosaic kit is compatible with cfDNA only.

We have successfully tested FFPE samples with between 10 and 80 ng gDNA input with the duet evoC assay and 20 and 80 ng gDNA for duet +modC. We followed the duet evoC/duet +modC user guide. PCR amplification cycles may need optimisation. We recommend 1 extra cycle PCR for all input ranges e.g. for <20g use 9 cycles, 20 to <40ng use 8 cycles, 40 to 80ng use 7 cycles:

*Note: the extra PCR cycles for FFPE samples are based on the gDNA PCR cycle recommendations.

See how duet evoC can identify DNA methylation markers from FFPE samples.

From our testing, samples with DIN (DNA Integrity Number) score of 1.6 or higher should be compatible with duet.

We have tested FFPE DNA samples with a range of DIN scores of 7.5, 6.5, 2.8, and 1.6 representing untreated, mild, moderate, and severe damage respectively. Lung cancer and CRC FFPE samples with DIN scores of 3.9 and 3.8 (moderate damage) were also tested. All performed as expected. For samples with moderate or severe damage (lower DIN scores), we observed slightly reduced yield, so we recommend adding one extra PCR cycle during library amplification for FFPE samples (see guidance above). See our FFPE technical note for full details on tested conditions and results.

No, chemical treatments such as dUTP/uracil-DNA glycosylase (UDG) are not applied prior to WGS library preparation to reduce the effect of DNA damage from formalin fixation, or needed to be used with biomodal evoC. biomodal evoC protocol uses USER enzyme to cleave at dUTP in our hairpin adapters. If dUTP was included in our DNA template it will also be excised by USER and not produce a final library.
The duet multiomics solution evoC employs a robust 6-base sequencing workflow that accurately distinguishes between canonical and modified cytosines, maintaining the integrity of C-to-T mutations and ensuring reliable mutation detection and comprehensive genomic and epigenomic profiling from challenging formalin-fixed, paraffin-embedded (FFPE) material. This workflow does not require dUTP/UDG treatment, as its unique chemistry and computational analysis help to overcome the challenges of formalin-induced DNA damage, delivering high data quality and reliability for both genetic and epigenetic analyses in FFPE samples. Incorporating dUTP/UDG would deviate from the validated duet multiomics solution evoC protocol and is unnecessary given the demonstrated robustness of the current approach.

For targeted assays, duet evoC and duet +modC can be used in conjunction with the Twist Alliance Pan-Cancer panel (1.5Mb) and the Human Methylome Panel (123Mb). Supplementary laboratory user guides are available on our customer documentation portal, to be used alongside the main duet evoC/+modC user guides. Read our poster for more information on targeted performance.

For targeted workflows, we recommend following the standard duet evoC/duet +modC user guides for spike-in control dilutions. Detailed dilution guidelines can be found in the section "spike-in control DNA preparation" of the user guides and reference tables provided below:

Yes, custom panels are in principles compatible with duet. The panels must be specifically designed for methylation assays and, as with all custom panels, optimisation is required.

The average fragment size should be around 250 bp (recommended range of 220-280 bp). 

cfDNA normally shows a prominent peak around 170 bp indicative of mononucleosomal DNA associated with one histone. Any subsequent nucleosome multimer peaks would be smaller and included in the region of 50-700 bp

Examples of gDNA contamination: 

• cfDNA contaminated with gDNA: trace shows a clear extra peak at >800bp (note that the sample conc. is low). 

• cfDNA contaminated with gDNA: trace shows smear above the nucleosome multimers and a small peak >800bp. 

The adapter sequences are TruSeq type. Sequences are below:

• Read 1: AGATCGGAAGAGCACACGTCTGAACTCCAGTCA.
• Read 2: AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT.

They can be found on the Illumina website under the TruSeq adapter reference page.

The hairpin sequence is AATGACGATGCGTTCGAGCATCGTCATT.

UDIs can be ordered with the core kit and are shipped in a separate box. However they should be shipped at the same time as the core kit. They are available in 3 different formats: 8, 24 (both supplied in tubes), and 96 UDIs (supplied in a 96 well plate). You can access the UDI sequences in Excel format on the biomodal customer documentation portal.

Note that duet UDIs 1C-8C (4103) are unique and differ from duet UDI 1-24/1B-24B(4102). duet UDIs 1-8 (4101) are identical to the first 8 UDIs in the duet 1-24/1B-24B kit. duet 1-24 and 1B-24B (4102) are identical.

Also, note that the 96 UDI format is unique and differs from the UDIs 1-8 or 1C-8C and UDI 1-24/1B-24B, except for three i5-only index sequences. If you are planning on combining the 96 UDIs kit with the 8 or 24 UDI kits, we recommend excluding the UDIs sharing overlapping i5 indexes. Please refer to the UDI sequence excel sheets on the documentation portal.

1 μl of supernatant can be saved for Qubit analysis in the event of low yields after the final library quantification stage for troubleshooting purposes.

For 80 ng input yield should be above 1 ng/μl and for 10 ng cfDNA input should be above 0.25 ng/μl.

Shrunken beads are fine to use. However, using beads after 3 months of unsealing the pouch is not recommended.

This is normal and it is ok to use.  

The iron in the reaction can sometimes turn the master mix to a pale pink. It is fine to proceed.

We recommend using the Diagenode magnetic rack when possible as we have found it to be the most reliable and we have optimised our assay with it.

We recommend 10 minutes of incubation time for optimal yield. Lower incubation time might impact yield.

We recommend to flick to mix to resuspend MT enzyme beads (not vortex as it is an enzyme). It is best practice to keep one tube per sample as pooling to re-aliquot might lead to a volume loss and not enough reagents for the last sample, impacting performance.

Beads can vary in shape due to the manufacturing process; this will not impact performance and all beads should be treated the same way.

It depends on the application; it's important to consider the specific questions you are asking of your data. We recommend 30x coverage to fully leverage the multimodal nature of the 6-base genome.

Lower sequencing depths may be suitable for certain applications or sample types, but this should be evaluated based on your specific needs. We recommend reaching out to our support team for further recommendations.

In collaboration with Element, we have validated with standard whole-genome workflow sequencing of duet evoC and duet +modC libraries on the Element AVITI24™ and AVITI™ sequencing instruments. Customers may follow the standard duet evoC or duet +modC User Guide up to the Sequencing section. At sequencing, Element‑specific parameters must be used:

• Loading concentration: start with 14.5 pM.

• PhiX: include 2% PhiX spike‑in. If running at 1× coverage, no PhiX spike‑in is required.

• Run configuration: 2 × 151 bp paired‑end sequencing, including 2 × 8 bp UDI index reads.

• Custom sequencing recipe: Element instruments require the EnhancedShortInsert custom recipe. Customers must contact their Element Field Applications Scientist to obtain it.

• Additional details: for full sequencing recommendations, see the supplementary laboratory user guide for sequencing on Element AVITI™ Platforms.

You can find a poster showing how duet evoC can generate accurately both epigenetic and genetic information using the Elevent Aviti sequencing platform. Note that for targeted workflows, some optimisation might be required.

We should avoid pooling libraries that have significantly different insert sizes (for example WGS and duet).

The optimal sequencing depth depends on your specific application and research goals. To fully leverage the multimodal nature of the 6-base genome, we recommend a depth of 30x coverage.

For a 30x whole human genome sample, the recommended read pairs are:

• 920M read pairs on NovaSeq 6000 or NextSeq 1000/2000, or
• 1050M read pairs on NovaSeq X

These estimates are based on a 2x151 paired-end whole genome duet run using duet +modC or duet evoC kits.

Yes, for best results, we recommend a loading concentration of 150 pM and adding 10% PhiX (with software 1.2 and below) or 5% PhiX (with software 1.3 and above) as a spike-in. To minimise demultiplexing issues, multiplex at least 4 samples and avoid mixing different sample types, as shorter insert sizes are preferentially amplified. For consistency, we recommend using the same sequencing platform across studies.

The pipeline outputs industry-standard, analysis-ready files (FASTQ, BAM, VCF, BedMethyl, CRAM) and comprehensive duet-specific data formats (cytosine report, Zarr, ASM, in addition to summary reports with quantitative metrics). Note that the pipeline output files vary slightly depending on the kit used. Details of the files generated can also be found in the data interpretation guide.

The pipeline generates standard file formats that can integrate with standard downstream analysis tools. This is covered in the biomodal data interpretation guide.

You should factor in approximately 2,000 CPU hours for 8 samples at 30x coverage.

Estimated timings are as below (note: these figures are intended as guidance estimate and are based on pipeline version 1.4.2. Actual resource requirements may vary depending on pipeline version and execution platforms):

1. Running in a cloud tenancy, allocating exactly the compute resources required at each stage: ~5 hours.
2. Running on a single dedicated host with 32 cores and 64 GB RAM: ~4 days.
3. Running on a single dedicated host with 64 cores and 64 GB RAM: ~2 days.
4. Using a Slurm cluster of 4 hosts, each with 32 cores and 64 GB RAM: ~1 day.

Yes, reference files are provided by default for the Twist Alliance Pan-Cancer and Human Methylome panels. However, for custom panels, users will needs to generate the required reference file themselves. See the targeted section of the biomodal cli for more information.

The kit is compatible with gDNA and cfDNA, and we have also successfully tested FFPE samples and targeted workflows. Additionally, customers can generate their own reference genome using our reference pipeline see the cli reference genome section.

duet sequencing data can be processed using biomodal’s duet pipeline. This pipeline processes the raw FASTQ files by resolving the hairpin constructs to obtain a consensus read of the native base on the original strand. It then performs trimming, alignment and annotation to produce analysis-ready outputs including BAM/CRAM, VCF, BedMethyl/BedGraph and ASM files. For more information or assistance with setup, check our software documentation hub.