Questions?

Orders and shipping.

How much do sequins cost?

Sequins are currently provided for free as part of an early-release trial scheme to users performing non-profit research. However, we do request users cover costs for the cryogenic shipment of RNA sequins (see below).

How do I order sequins?

To order sequins, you have to complete the registration form here https://www.sequinstandards.com/register-user/

Please note that these terms and conditions must be completed by an intuitional official that is authorized to make legal commitments on behalf of the recipient organization. Once your form has been received and your details have been confirmed, we will ship sequins to the recipient address. If you would like the sequins to be shipped to a different address, please indicate. We will notify you by email when the sequins have been dispatched.

Can sequins be used in commercial for-profit applications?

Yes, sequins can be used to test and develop commercial products, and in commercial applications. If you would like further details, please contact us at sequin@garvan.org.au.

How are DNA sequins shipped and what are costs?

DNA sequins are shipped in lyophilized form at room temperature with TNT express shipping (www.tnt.com). Once DNA sequins have dispatched, we will notify you with an email and a tracking ID. The shipment should take less than ~2 weeks to arrive, but can depend on your location.

We do not charge any costs for shipping DNA sequins.

How are RNA sequins shipped?

RNA sequins are shipped frozen.

Within Australia and United States, RNA sequins are shipped on dry ice with FedEx, and can cost up to ~$200USD. We will confirm payment details (or FedEx account number), a cost estimate, and shipping details prior to dispatch.

Internationally, we ship RNA sequins within a liquid nitrogen dewar (with Cryoport). Shipment typically costs $500-$700USD, depending on the final destination. We will confirm payment details, a cost estimate, and shipping details prior to dispatch.

How long will my sequins take to arrive?

Sequins should arrive within 2 weeks of your order, although this can depend on your location. We will confirm the dispatch of you sequings with an email, and issue a tracking ID so you can monitor the shipment.

If you experience an unexpected delay in receiving your sequins, please contact us on sequin@garvan.org.au.

How much/many sequins do I receive?

We typically ship two vials of sequins, with each tube containing either 250ng of DNA sequins (genome and metagenome) or 150ng of RNA sequins.

How much sequins you require depends on the input requirements of your library preparation protocol. For example, the TruSeq DNA Nano Library Prep requires a sample input of 100ng of genomic DNA. Therefore, if you are adding 1ng of sequins to your sample (ie. at 1% concentration), then a single vial is should be sufficient for 250 reactions.

When you place your order, please let us know if you need additional sequin vials for your project. If you are unsure, we have experience using sequins in a wide range of different library preparation and sequencing protocols and will be happy to advise you.

How should I store sequins?

DNA sequins are shipped in lyophilized form at room temperature. We suggest that they are stored (whether in lyophilized or resuspended form) at  –20°C, where they are stable for at least 24 months.

RNA sequins should always remain frozen until they are thawed for use. We recommend that RNA sequins are stored at -80°C for up to a year.

Sequin use.

How big are sequins?

Sequins come in different sizes, but are typically between 2kb and 10kb in length.

What sequencing technologies are sequins compatible with?

Sequins are simply RNA and DNA molecules, and are compatible with almost all sequencing technologies (indeed, we often use sequins to compare and benchmark different technologies).

We have successfully used sequins with different sequencing platforms (Illumina, PacBio, Oxford Nanopore etc.), library preparation methods (KAPA, Illumina, PCR-based, PCR-free etc.) and applications (whole-genome sequencing, targeted sequencing, RNAseq etc.).

If you have any question about whether sequins can be used with your sequencing application, please contact us and will be happy to advise you.

How much sequins should I add to my sample?

Using sequins with non-targeted sequencing applications (such as WGS, RNAseq, metagenomics) is relatively straightforward; the fraction of reads in the output library derived from sequins will reflect the fractional abundance at which sequins were originally added to the user’s DNA sample.

We typically recommend that sequins are added at ~2% concentration relative to the sample. For RNA sequencing and metagenome applications, this will achieve sufficient coverage of sequins for analysis, whilst minimizing the fraction of reads sacrificed to spike-in standards. For WGS applications, this is sufficient to achieve matched sequencing coverage with the accompanying diploid human genome. Please refer to the laboratory protocols for specific guidelines on how much the sequins should be diluted and added to achieve this fractional concentration.

Sequins are typically added at a lower concentrations to targeted sequencing approaches (often as low as 0.1%). However, the exact amount of sequins to add will depend on both the amount of sequins and accompanying human sample that are targeted for enrichment. We recommend that users working with custom capture/amplicon panels should validate the suggested spike-in amounts, as these values may need to be adjusted depending on their panel design and library preparation protocol. Care must be taken not to add sequins to the sample in excessive stoichiometry, since exponential amplification can then lead sequins to over-populate the sequenced library.

The prevalence of sequins in a target-enriched library preparation can also be affected by additional variables such as DNA sample quality, library complexity and PCR amplification. To mitigate risk, we recommend users validate library composition using qPCR prior to sequencing. Further information on estimating the dilution factor and the validation of libraries with qPCR is provided in: Blackburn et. al., (2019) Nature Protocols.

 

Can sequins be used with non-human organisms?

Yes, whilst sequins have been largely designed to represent human genetic features, they can still be used with RNA or DNA extracted from any organism. We have successfully used sequins with mouse, zebrafish, arabidopsis RNA and DNA samples.

Please note that when using the sequins with non-human organisms, you may prefer to add the decoy chromosomes to your organisms reference genome index prior to alignment (see Software Documentation for further details).

Are sequins compatible with targeted/gene panel/exome sequencing?

Yes, sequins are compatible with targeted sequencing using probe hybridization and enrichment (e.g., gene panels and exome sequencing). Indeed, sequins can evaluate the errors and biases introduced during target enrichment, which form a dominant source of technical variation that confounds accurate diagnosis.

When using sequins with targeted sequencing, additional probes must be included to target and enrich sequins, alongside the target genes in the accompanying sample. Given sequin and sample sequences do not compete for the same capture probes, the capture of sequins does not impact the capture of target human sequences. Ideally, the additional probes that target sequins should mirror the sequences of probes that capture corresponding human genes. This allows the capture of sequins to mirror the capture of human genes, thereby providing a highly effective internal control that captures the same errors introduced during hybridisation and enrichment.

Please note that care must be taken to not add to much sequins to the sample prior to capture so as not to over-populate the final library. Due to the enrichment of targeted sequins, only a minimal amount of sequins is often enough to achieve sufficient coverage. The exact amount of sequins to add will depend on the collective size of captured genome regions and sequin sequences. Additional variables, including DNA sample quality, library complexity and PCR amplification can also impact the enrichment of sequins. We encourage users to incorporate sequins thoughtfully into their designs, targeting only those standards that will provide relevant information for their specific project, and validate library composition using qPCR prior to sequencing when using a new capture panel designs.

We have successfully used sequins in many different RNA and DNA target-enrichment experiments. If you have any question about how to use sequins with a sequencing application, please contact us and will be happy to advise you.

Can sequins be used with amplicon-based sequencing?

Sequins are compatible with multiplexed PCR strategies that amplify genome sequences of interest prior to sequencing. However, integrating sequins with these approaches requires additional primers to amplify sequins along with the target RNA or DNA sequences. Ideally, the primers used to amplify sequins should mirror the primers used to amplify corresponding target RNA or DNA sequences. This allows the amplification of sequins to mirror the amplification of target RNA or DNA sequences, whilst avoiding primer sequence competition and dimerization.

Care must be taken not to add too many sequins to the sample prior to amplification, since exponential amplification can then lead sequins to over-populate the sequenced library. Therefore, we recommend that users should validate the amount of sequins spiked-in to samples according to their amplicon-based enrichment design, protocol and needs. Users may also validate library composition using qPCR prior to sequencing.

Can sequins be used with FFPE or damaged samples?

We have successfully tested sequins in comparison with a range different sample types, including fresh, frozen and formalin-fixed, paraffin-embedded (FFPE) samples.

However, additional caution should be exercised when using sequins with DNA samples of poor or degraded quality, where sequins may undergo library construction preferentially at the expense of accompanying DNA sample. This limitation can be offset by adding sequins at a lower fractional abundance and/or using qPCR to validate that sequins do not over-populate the constructed library.

If you are unsure or concerned, please contact us and we are happy to advise.

What is the difference between the 'genome' and 'matched' sequin mixture? Which should I use?

For human genome sequencing, we typically provide two alternative sequin mixtures labelled; genome and matched

If you are sequencing only a single genome DNA sample, we recommend you use the genome mixture.

However, if you plan to sequence both a tumor and a matched normal sample for cancer applications, we recommend you use either of the alternative sequin mixtures. The genome mixture contains somatic cancer mutations as well as germline variants, and should be added to the tumor DNA sample.  Whilst the normal mixture contains only germline variants and should be added to the matched normal DNA sample. This normal sequin mixture thereby provides an un-mutated background sequence, against which somatic mutations in the tumor mixture can be identified.

Do you have a question that is not answered here? Send an email to sequin@garvan.org.au