Synthetic Controls

All 456 variants are stored in one tube.

The Twist cfDNA controls are double stranded DNA. They are a mixture  of synthetic “ctDNA” molecules designed and printed with Twist technology and spiked into the background cfDNA to form each specific VAF level.

The standard contains many difficult-to-detect variants, including structural variants and INDELS ranging from 1 bp to 30 bp. Detection of individual variants can depend on what aligner and alignment setting the customer is using. These hard to detect variants are for experienced customers using their pipeline. So just having an understanding of it. SNVs and small INDELs (e.g. 5 bp or smaller) are more straightforward for customers to detect.

The Twist background cfDNA high-fidelity non-amplified DNA is derived from our proprietary fragmentation  process, and we have observed an extremely low mutation rate compared to competitor material.

Once prepared into a library with indices, there are no issues with pooling this control with patient samples for simultaneous sequencing.

Twist performed a literature search to arrive at a list of curated, clinically-relevant (i.e. actionable) cancer-associated variants, as well as using the COSMIC database as a reference, where matching entries are available It includes the many prevalent variants in adult solid tumors. The 456 variants occur in 85 unique genes. Twist also separately annotates   the set of clinical relevant variants (>140 of variants).

The configuration of variants and their precise reference and alternative sequence are listed on the Twist website.

Twist runs target enrichment NGS on the 0% and 5% VAF to ensure non-detection and detection, respectively, at all variant sites. Additionally, six sites across all VAF levels are confirmed via ddPCR.

Twist performs whole exome sequencing for the background cfDNA background and utilizes a target capture on the 5% material, in addition to a ddPCR on the intermediate VAFs.

Twist recommends using "Low-EDTA TE," which is 10 mM Tris pH 8, 0.1 mM EDTA. If EDTA must be avoided for compatibility with downstream steps, Qiagen Elution Buffer (10 mM Tris-HCl, pH 8) can be used.

This deletion occurs in the gene ATM at a genomic context with 10 consecutive Ts. These consecutive Ts makes the locus a "slippery" sequence, where the DNA polymerase replicating a copy of the genome can have the template strand slip relative to the strand being synthesized, leading to an INDEL.

The same effect has a high likelihood of occuring by the same mechanism when a PCR polymerase copies the sequence during library prep PCR. Thus, this locus is especially likely to spontaneously form the variant sequence due to PCR cycling during ordinary library prep.

When the allele is not present at all, any human genomic sample (including the Pan-cancer Reference Standard) will show an elevated VAF for this mutation due to PCR error. (In pan-cancer 0% VAF, it shows up at a higher VAF than 99.8% of other variant sites in the standard.) Thus, the background noise is higher due to the genomic context, meaning the Limit of Blank (LoB) of this variant is likely higher than other loci due to the sequence contest.

When the allele is present, the true allele frequency will be added “on top of” the noise level of the locus, meaning this variant will likely have a higher Limit of Detection (LoD) than other variant sites.

Using UMI adapters (like the Twist UMI Adapters) and running consensus collapse and/or duplex collapse can help decrease the noise at this locus due to PCR error in library prep.

All 456 variants are confirmed by NGS (for both 0% and 5% VAF), and the 0.1%, 0.25%, 0.5%, 1% and 2% are generated from a blending of 0% and 5% stocks. We selected a set of six representative and relevant variants  across 0% to 5% VAFs for ddPCR, showing quantitative formulations and a continuous serial dilution.

The full kit contains 7. If an individual tube is 3 ug, it is a single tube. The 2 tubes 300 ng configuration has 2 tubes.

Twist recommends starting with 30 ng of starting input. 20-30 ng is a typical range for liquid biopsy assay.

The variant fragments are blunt-ended, double-strand DNA molecules.

Twist has tested the material to be a suitable standard in non-fragmenting end-repair and A-tail ligation (ERAT) library preparation protocols, as well as in amplicon protocols, such as ddPCR and qPCR. The performance of the v2 Pan-cancer Reference Standard is similar to the v1, and to any other dsDNA input into library prep.

Cell-free DNA is generated in a highly specific biological process that yields a characteristic fragment length profile. Thus, we recommend preparing a library that does not change the size profile in any way: using the "Twist Mechanical Fragmentation Library Prep Kit" without any additional fragmentation treatment. Our library insert sizes look superb when we use that method. We have used Twist UMI adapters extensively on the cfDNA samples, and they work great for duplex consensus and getting good assessments of the mutation rates.

The Twist wild-type background cfDNA mimic is cell-line-derived and treated with our proprietary method for closely mimicking the fragmentation profile of naturally occurring cfDNA, which is characterized by a sharp mononucleosomal peak and a secondary dinucleosomal peak.

The Twist cfDNA is sourced from an immortalized cell line, which enables us to create large production lots  with consistent lot to lot genotypes and performance.

Twist's SARS-CoV-2 RNA controls and Panel are for research use only.

10mM Tris-HCl and 0.1mM EDTA pH 8.0

The shelf life of the RNA controls is 24 months from the date of manufacture. 

Our mpox virus controls are not ISO certified. To respond quickly to the public health need, we decided to launch the controls without an ISO certification.  However, we are open to getting these SKUs ISO certified.  Please let us know if you are interested in ISO certification.

Twist provides a suite of research tools and NGS products, including custom controls

and reference standards for mpox, respiratory diseases including coronaviruses,

influenza viruses, paramyxoviruses and enteroviruses as well as liquid biopsy and cancers. 

If your product of interest is not included in our offering, like panels or controls, please contact your sales manager or customer support at customersupport@twistbioscience.com.  We will help you with potential custom solutions.

The Twist Synthetic human mpox controls have a concentration within 50% to 200% of the targeted 100,000 copies/ul as determined by ddPCR (T1 assay), or between 50,000 to 200,000 copies/µL.

The Certificate of Analysis document reports out at least 100,000copies/µL fragments concentration with a range of 25% to 400%, or 25,000 - 400,000 copies/µL per fragments.

Each of the mpox controls contains 100 µL at 1x10^5 copies/ µL fragments.

The mpox sequences are to be provided upon request. Contact customersupport@twistbioscience.com for inquiries.

Twist Synthetic hMPXV Control 1 (EPI_ISL_13056236) 106051 (component) / 106056 (finished good)

Twist Synthetic hMPXV Control 2 (NCBI:ON585037) 106055 (component) / 106059 (finished good)

Each control covers 80-85% of the mpox virus genome.

The mpox controls are validated and they are compatible with CDC protocols. The CDC protocols can be found here.

We recommend starting with 1 uL per reaction and adjusting usage based on the results.

The controls are shipped frozen in dry ice. Storage temperature is recommended at -80°C, but the controls can be stored at -20°C for short term storage.

There are two Clades: Congo Basin Clade (Control 1) 

West Africa Clade (Control 2)  

Control 1 contains 154 fragments while Control 2 contains 151 fragments. Each of the fragments are around 1.8kb.

Since the mpox controls are double-stranded DNA controls, any purchase outside the US requires an export license due to the biosecurity concern. For US-based shipping, an export license will not be needed but please work with your NGS Account Manager to generate a quote and place an order.  If you are based outside of the US, please contact Twist customer support (customersupport@twistbioscience.com)  and provide your name and institution’s name and address.  Our customer support team will work with you to get your export license granted.

Repetitive sequences and areas of extremely high or low GC content as well as homopolymers  are excluded in the genome.  It’s challenging to target those regions.