Between 1993 and 2018^[1] breast cancer rates in women increased by 24%, and in the UK in 2022, breast cancer was the most common cause of death for women between 35-49 and 50-64 years of age.^[2]

We are delighted to see Isohelix products used for Breast Cancer research, with two recent UK-based studies using GeneFix Saliva collection devices to collect and stabilize DNA in saliva samples collected from study participants:

• BRCA Direct: A Digital Pathway for BRCA-Testing in Breast Cancer
• Breast Cancer Risk Assessment in Younger Women (BCAN-RAY) study

GeneFix Saliva Collection Products

The GeneFix™ range of DNA/RNA products has been designed to maximize the yields and purity of DNA/RNA collected and stabilized from saliva. GeneFix kits are ideal for collecting samples from study participants at home or in the clinic, as they are non-toxic, simple to use, and contain a reagent that stabilizes DNA at room temperature for up to 60 months. After sample collection, kits can be mailed to the lab for DNA extraction.
Read on to learn about recent studies using GeneFix products for Breast Cancer Research.

BRCA-Direct: A Digital Pathway for BRCA-Testing in Breast Cancer

The BRCA-DIRECT study was funded by Cancer Research UK and affiliated with the Institute of Cancer Research, SHORE-C,  Manchester University Foundation Trust, and The Royal Marsden Foundation Trust. The study aimed to provide an easy way for patients with breast cancer to access genetic testing within the NHS.

The BRCA1, BRCA2, and PALB2 genes are associated with hereditary breast cancer, so Identification of a pathogenic variant in one of these genes can have health implications for patients and their relatives. This study examined the feasibility, safety, and acceptability of a digital information model.

Participants provided a saliva or blood sample and accessed a digital platform using a device connected to the internet.  Family history details were collected, and information was gathered about the general knowledge of BRCA testing. Participants were asked about their anxiety levels at different points in the process. Half of all those who took part saw digital information, and half booked a standard appointment with genetic counselors.

Participants were then randomized to receive their results digitally or by booking a telephone appointment with a genetic counselor. Everyone who had a positive result was then referred to their local clinical genetics team. If the digital pathway is successful, the concept could be expanded to other cancers and hospitals.

​We are looking forward to seeing the results of the study that has led to further funding for a pilot across North Thames GLH, funding by SBRI Healthcare/NHS England Cancer Programme: SBRI Healthcare – NHS Cancer Programme awards £12.1 million to accelerate new front-line innovations that detect and diagnose cancer earlier

The BCAN-RAY study

The Breast Cancer Risk Assessment in Younger Women (BCAN-RAY) study began in May 2023. BCAN-RAY is one of the first research studies in the world to identify new ways to predict the risk of younger women getting breast cancer. The study aims to evaluate a comprehensive breast cancer risk assessment strategy among a diverse ethnic and socioeconomic population of women aged 30–39 years without a strong family history of breast cancer.

Two hundred fifty women previously diagnosed with breast cancer without a strong family history of the disease will be studied alongside 750 women (control participants) in the same age group who have not had breast cancer and who also have no strong family history of the disease. 

Control participants will complete questionnaires about breast cancer risk factors, undergo low-dose mammograms, and donate a saliva sample, which is collected using Isohelix GeneFix Saliva Collection Kits. Saliva samples will be used to analyze the genetic makeup of participants and identify those at higher risk using a tool called a polygenic risk score, which is a powerful predictor of breast cancer risk.

The BCAN-RAY study should complete recruitment in May 2025, and we look forward to seeing the results.

The study is funded by Cancer Research UK via the International Alliance for Cancer Early Detection (ACED), The Christie Charity, and The Shine Bright Foundation. It is led by Manchester University NHS Foundation Trust, and delivered at The Nightingale Centre at Wythenshawe Hospital and breast oncology centers across Greater Manchester and Cheshire.

Please follow the link to the Sarah Harding Breast Cancer Appeal: A letter from Girls Aloud (christie.nhs.uk) 

Read our blog article about genomic analysis using saliva instead of blood: https://isohelix.com/saliva-instead-of-blood/

Find out how to simplify Saliva and Buccal Swab sample collection and processing: https://isohelix.com/how-to-simplify-saliva-and-buccal-swab-sample-collection-and-processing/

Click here to find out more about our GeneFix Saliva Collection Products: https://isohelix.com/genefix-saliva-dna-rna-collection/

References

[1] Facts and figures | Breast Cancer UK

[2] Office for National Statistics. Deaths registered in England and Wales: 2022-2023

Long read sequencing was described as the “method of the year[i]” In an article published in Nature Methods at the start of 2023. The development of long read sequencing has significantly expanded the possibilities for genomic analysis.

Despite challenges such as the cost and the complexity of data analysis, the technology continues to improve, with increased accuracy, affordability, and accessibility.

This article introduces long read sequencing, highlights some of the key advantages compared with short read sequencing, and gives some key applications for long read sequencing.

Key points:

• Long read sequencing length
• Long read sequencing methods
• Advantages of long read sequencing vs short read sequencing
• Challenges with long read sequencing
• Long read sequencing technology platforms
• DNA extraction for long read sequencing
• Should I use short read or long read sequencing?

Long Read Sequencing Length

Long-read sequencing, sometimes called “third generation sequencing,”  is a DNA sequencing technique that enables the sequencing of much longer stretches of DNA, typically ranging from thousands to over a million base pairs. By comparison, traditional short read sequencing typically captures sequences of 100-500 base pairs.

Long Read Sequencing Methods

Long read sequencing can be either “true long read” sequencing, or ‘synthetic long read sequencing.”

“True long read” sequencing directly reads longer fragments of DNA, typically ranging from thousands to over a million base pairs. This method is employed by companies such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), enabling the sequencing of long DNA strands in a single continuous process.

“Synthetic” long read sequencing uses short read sequencing data to reconstruct longer stretches of DNA. This method is employed by companies such as, Element Biosciences and Illumina (primarily known for short read sequencing). In synthetic short read sequencing, short DNA fragments are barcoded and sequenced using standard short read technology platforms, and computational methods are then used to assemble the short reads into longer sequences based on barcodes and overlaps, effectively creating a “synthetic” long read. This approach provides some of the benefits of “true” long read sequencing, like the better assembly of repetitive regions or complex genomic structures, without needing specialized long read sequencing equipment.

Both long-read and short-read sequencing have pros and cons. A “hybrid” DNA sequencing approach combines different DNA sequencing technologies to leverage each other’s strengths while compensating for their weaknesses. This approach typically involves using both long-read and short-read sequencing technologies together.

Advantages of Long-Read Sequencing vs Short Read Sequencing

The development of long read sequencing has been driven by the search for more complete and accurate genomic information.

The key limitation of short read sequencing is the inability to sequence long stretches of DNA. If the sequence of a large region of DNA is required, e.g. for a genome assembly, then the DNA has to be first fragmented, then amplified and sequenced. Bioinformatics tools are used to assemble these short sequences to give the full length sequence. However, if there is insufficient overlap between these shorter DNA fragments, there will be gaps or errors in the final sequence. Also amplification steps can introduce sequencing errors, particularly in repetitive regions of the genome.

The key limitation of short read sequencing is the inability to sequence long stretches of DNA. If the sequence of a large region of DNA is required, e.g. for a genome assembly, then the DNA has to be first fragmented, then amplified and sequenced. Bioinformatics tools are used to assemble these short sequences to give the full length sequence. However, if there is insufficient overlap between these shorter DNA fragments, there will be gaps or errors in the final sequence. Also amplification steps can introduce sequencing errors, particularly in repetitive regions of the genome.

Long read sequencing can provide a more comprehensive view of a genome than short read sequencing, enabling better identification of structural variants and repetitive regions that are often challenging to resolve with short reads, because it is difficult to reassemble sequencing data over long stretches of DNA.

Long reads can be particularly useful when:

1. Resolving Complex Genomic Regions: Long reads are particularly advantageous when sequencing regions with repetitive elements, structural variants, and complex rearrangements, which are often challenging for short-read technologies.
2. Assembling Genomes: Long-read sequencing provides more contiguous and accurate genome assemblies. This is especially important for de novo sequencing, where a reference genome is not available.
3. Detecting Structural Variants: Long-read sequencing is useful for detecting large structural variants such as insertions, deletions, inversions, and translocations, which play significant roles in genetic diversity and disease.
4. Phasing and Haplotyping: Long reads can span entire genes or large genomic regions, allowing for the accurate phasing of alleles and haplotype reconstruction.

Challenges with Long Read Sequencing

Despite its advantages, long-read sequencing historically faced several challenges, including higher costs and error rates compared to short-read sequencing. However, ongoing technological advancements are rapidly addressing these issues.

1. Error Rates: Long read sequencing historically had higher error rates compared to short reads, affecting data accuracy. However, read accuracy is improving.
2. Cost: The initial cost of long read sequencing technologies and associated data analysis can be higher than short read sequencing.
3. Bioinformatics: Analyzing long read data may require specialized bioinformatics tools and computational resources, due to the unique characteristics of long reads. Data processing can take longer than with short read sequencing.

Long-Read Sequencing Technology Platforms

There are several platforms that facilitate long-read sequencing, with the two most well known being Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT).

Pacific Biosciences (PacBio): PacBio’s Single Molecule Real-Time (SMRT) sequencing technology can generate reads averaging 10,000-15,000 bp. The technology utilizes real-time observation of DNA synthesis, where fluorescently labelled nucleotides are incorporated by DNA polymerase, allowing for the continuous reading of the sequence.

Oxford Nanopore Technologies (ONT): ONT’s nanopore sequencing passes a DNA molecules through a nanopore embedded in a membrane. As the DNA translocates through the pore, changes in ionic current are measured and translated into sequence data. ONT platforms can produce ultra-long reads offering unparalleled length and flexibility.

DNA Extraction for Long Read Sequencing

Short read sequencing generally requires DNA fragments between 100 and 600 base pairs in length, and therefore, it can tolerate somewhat degraded DNA since the required fragment size is smaller. DNA is often fragmented mechanically (using sonication) or enzymatically during sample preparation.

Short read sequencing generally requires DNA fragments between 100 and 600 base pairs in length, and therefore, it can tolerate somewhat degraded DNA since the required fragment size is smaller. DNA is often fragmented mechanically (using sonication) or enzymatically during sample preparation.

Long-read sequencing requires high-quality, high-molecular-weight DNA, typically upwards of 10,000 base pairs. Any nicks or breaks in DNA strands can significantly impact the ability to generate long reads. The main limiting factor for ONT read lengths is the DNA extraction; Jain et al (2018) found that read lengths produced by the MinION [iii] nano pore sequencer were dependent on the input fragment length[ii]. This often necessitates more careful handling and extraction procedures, and there are several extraction methods and commercially available kits for preparing DNA for long read sequencing.

Should I use Short Read or Long Read Sequencing?

The choice between long-read and short-read sequencing methods will depend on the specific requirements of the research question including the characteristics of the region to be amplified, sample type, cost, and accuracy.

Recent improvements in error correction algorithms, cost reduction strategies, and hybrid sequencing approaches that combine long-read and short-read data are paving the way for broader adoption.

[i] Marx, V. Method of the year: long-read sequencing. Nat Methods 20, 6–11 (2023). https://doi.org/10.1038/s41592-022-01730-w

[ii] Jain M et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat.  Biotechnol 36, 338–345 (2018).

[iii] MinION is a trademark of Oxford Nanopore

This article explains how to optimize and streamline sample collection and processing for nucleic acid analysis, to ensure consistent and reliable results while minimizing sample collection, processing, and shipping costs.

Key things to consider are:

• Sample collection protocols
• Nucleic acid stabilization
• Sample transport
• Streamlined sample processing
• Sample tracking

Use Simple Saliva Sample Collection Protocols

Buccal swabs and saliva samples can be collected non-invasively, which encourages more people to participate in studies. This is especially important for large-scale population studies, where broad participation is crucial for the validity of the results.

Allowing donors to collect samples at home removes the need to travel to a clinic or laboratory, which is particularly beneficial for individuals with mobility issues, busy schedules, or living in remote areas. Collecting samples at home can significantly reduce costs associated with sample collection by eliminating the need for dedicated space and staff to handle sample collection in a clinical setting.

Isohelix sample collection kits include several features that simplify sample collection:

• All kits include clear instructions for use that can be followed by donors at home
• Isohelix GeneFix saliva collection kits have a unique funnel design that prevents spillages or buffer flow back.
• GeneFix™ collection tubes have clear, legible markings to indicate the correct volume of saliva to be collected
• Buccal Swabs are provided in easy-to-handle formats

Nucleic Acid Stabilization

Isohelix offers several options for nucleic acid stabilization that remove the need for a cold chain during sample shipment, significantly reducing the costs and logistical challenges associated with sample storage and transport.

Stabilization Reagents

Including collection tubes prefilled with a nucleic acid stabilization reagent is a simple method of immediately stabilizing samples at the point of collection, with minimal handling steps and cross-contamination risk.

For swabs and saliva collection, Isohelix collection tubes are pre-filled with BuccalFix or GeneFix stabilizing buffers that completely inhibit all enzymatic and microbial activity following sampling. As a result, DNA is fully stabilized, and the structure and integrity of the DNA are maintained. DNA can be stored in BuccalFix at room temperature for over 2 years and in GeneFix for up to 5 years.

Nucleic Acid Stabilization via sample desiccation

For shorter-term storage, RapiDri™ Swab kits (RD-01) include a microporous moisture-wicking pouch that rapidly dries the swab, stabilizing the DNA on the swab matrix, where it is protected from degradation. DNA can be stabilized for 3 months at ambient temperature, allowing safe and easy sample transport and storage.

Alternatively, Isohelix Dri-Capsules are silica gel capsules that offer an easy-to-use alternative to chemical stabilization for the long-term stability of buccal DNA on a swab head before isolation. DNA stabilized using Dri-Capsules is stable for 3 years at ambient temperature. 

Streamline Sample Transport

If collection tubes and sample packaging are sufficiently robust, stabilized samples can be sent to the laboratory for processing using regular mail, a straightforward and cost-effective method of sample transport.

There are a range of products available from Isohelix to simplify shipping:

• Isohelix collection tubes comply with UN3373 on leak-proof sample transport that specify that collection tubes must withstand defined physical pressures of 95KPA. 
• Mailing boxes are suitable for GeneFix Saliva collectors and have space to fit a transport pack underneath. They are designed to fit through most letterboxes and are certified to UN3373. They include a security sealing strip.
• Transport packs are suitable for saliva and swab samples and include an absorbent pad to contain leakages, a separate document pocket, and are tested to resist 95kPa.
• Saliva transport packs are specifically designed for saliva samples, featuring a slimmer profile for easier postage

Medium and High-Throughput Sample Processing

For large numbers of samples, automated systems reduce handling errors, save hands-on time, and increase processing throughput. Genefix High Throughput tubes (GFXH) for saliva collection have a key-fitting cap and locking base designed to fit automated de-capping machines. For buccal swabs, SwabCatcher tubes include a unique cap design that automatically removes swabs and is suitable for automated and high-throughput systems.

Isohelix also offers magnetic bead-based nucleic extraction kits, Buccal-Mag, and Saliva-Mag. These kits can be used manually or with most openly programmable automated DNA isolation and liquid handling systems fitted with magnetic racks and heat blocks.

Alternatively, for saliva collection, Isohelix has adapted the Saliva-Prep2 kit for use with 1.2ml or 2.0ml 96 – Deep Well Plates. This gives a manual method for isolating 96 samples per plate, requiring only low-cost, widely available benchtop laboratory equipment.

Sample Tracking

Easy and accurate sample tracking is a key component of sample collection and isolation. All GeneFix Saliva Collection tubes include a unique 10-digit, alphanumeric 1D barcode on the side of the tube which allows for full traceability of the sample from production to distribution and testing. Tubes can be specified with a 2D barcode on the base of the tube that corresponds with the 1D barcode on the side. Having a 2D barcode available on the base of the tube allows automation systems to scan the barcodes while in racking to maintain traceability when using high-throughput methods. Barcodes printed in triplicate on cryogenic labels are also available for attaching to tubes or paper. All barcodes are scannable on common readers, including those widely available on mobile phones, so that even patients at home can scan their devices to provide additional traceability.

Summary

By implementing simple standardized collection methods, and optimizing processing and shipping, the integrity and reliability of saliva and buccal swab samples can be preserved, ensuring the best possible nucleic acid analysis results whilst minimizing time and cost.

To find out more about how Isohelix products simplify sample collection, CLICK HERE

We were delighted to see that two recent, high-profile studies looking at the effects of spaceflight on astronauts’ microbiomes chose Isohelix buccal swabs to collect their valuable samples.

In the Nature paper, “Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight,” published by Tierney et. al., (2024), the team used Isohelix swabs to collect samples from three microbial ecosystems (oral, nasal, and skin), before and after a 3-day mission to space.

Using paired metagenomics and metatranscriptomics alongside single-nuclei immune cell profiling, the team characterized microbiomes before, during and after spaceflight. The study revealed shifts in the microbiome and immune response during short term spaceflight; although most microbiome alterations were transient, longer-term shifts were observed in the astronaut’s oral microbiomes.

Another Nature paper, “Spatial multi-omics of human skin reveals KRAS and inflammatory responses to spaceflight”, Park et al (2024), also looked at the effects of short-duration spaceflight on the skin. The study applied a wide range of -omics methods, and as part of this study, Isohelix swabs were used to collect samples of astronauts’ skin microbiomes. Metagenomics and metatranscriptomics analyses were then performed on the skin swab samples. A key finding was that post-flight samples showed significant up-regulation of genes related to inflammation and KRAS signalling across all skin regions.

The results from these two important studies can help guide spacecraft design and response countermeasures for astronauts on future missions, and we are excited to see Isohelix products being used to expand our knowledge in this area.

Isohelix swabs feature a unique swab matrix and quick-release surface that maximises nucleic acid yields. Swab batches are ethylene oxide treated and routinely tested for human DNA contamination, making them ideal for capturing precious nucleic acid samples.

CLICK HERE TO FIND OUT MORE ABOUT ISOHELIX BUCCAL SWABS FOR NUCLEIC ACID COLLECTION OF ORAL AND SKIN MICROBIOME SAMPLES

Isohelix also offer stabilization reagents for buccal swabs that offer long term sample stability prior to isolation

Link : Isohelix™ DNA Buccal Swab Stabilization – Isohelix

The body of research using Isohelix products is growing all the time. One that caught our attention recently was, “Exploring the legacy of African and Indigenous Caribbean admixture in Puerto Rico” by Taiye Winful et al. (2023).

The goal of this study was to assess the biogeographic origins of African descendant Puerto Ricans and to investigate the potential for Indigenous ancestry within this community.

2ml saliva samples were collected from self-identified African descendant Puerto Ricans residing in Puerto Rico using Isohelix Genefix™ collection kits. GeneFix kits are ideal for collecting samples from study participants in remote communities as they are non-toxic, simple to use, and contain a reagent that stabilizes DNA at room temperature for up to 60 months. After sample collection, kits can be mailed to the lab for DNA extraction.

When the samples reached the lab, DNA was extracted using the Isohelix Genefix Saliva-Prep 2 DNA kit, which uses a precipitation method to isolate high yields of pure DNA. The mitochondrial genomes of these individuals were sequenced, and Y chromosome haplogroups were genotyped for samples donated from males. Summary statistics, comparative analyses, and network analysis were used to assess diversity and variation in haplogroup distribution between the samples and comparative populations.

Results from the mitochondrial haplogroups showed that 66% had African, 5% had European, and 29% had Indigenous American matrilines. The Y chromosome analysis showed that 52% had African, 28% had Western European, 16% had Eurasian, and 4% had Indigenous American patrilines.

This study successfully used Isohelix GeneFix and Saliva-Prep kits to collect and extract DNA from saliva samples from study participants across Puerto Rico. The genomic analysis of these saliva samples have contributed to understanding how African descendants and Indigenous American admixture have shaped present-day communities.

CONTACT US to find out how we can help with your sample collection and DNA extraction needs.

The quality of DNA samples affects the quality of data produced by genomic analyses, and as analysis techniques become more sophisticated, DNA quality becomes even more important. For example, long-read sequencing enables the detection of variants that are often difficult to resolve with short reads, but it requires high-quality, high-molecular-weight input DNA to be successful.

DNA integrity must be maintained from the moment of sample collection to when the sample is processed in the lab to produce the best results. This can be particularly challenging with large genetic studies where DNA samples are collected from donors at home. Samples may be in transit for several days and exposed to wide temperature fluctuations before they reach the lab. DNA samples are particularly vulnerable to degradation in hotter, humid conditions.

The DNA integrity of a sample can be reduced during shipping and storage via several mechanisms :

• Enzymes (e.g. nucleases) and chemicals present in samples may degrade DNA
• Repeated freeze-thaw cycles, particularly if samples are exposed to low temperatures during shipping, may shear long pieces of DNA into shorter fragments
• Microbes present in samples may grow, adding to the proportion of microbial DNA present, and also producing chemicals and enzymes that may damage target DNA.

Microbial DNA present in samples can be a particular problem for microbiome analysis. During transport and storage, some microorganisms in the sample may continue to grow at the expense of others, meaning that the sample is no longer representative of the original community.

Traditionally, freezing was the standard method for DNA preservation, but freezing presents logistical challenges, especially in field settings or when samples need to be transported over long distances. Freezing requires a lot of energy, the cold chain must be maintained, and the weight of the ice used to keep samples frozen adds to shipping costs. If the cold chain fails during shipment or storage and the sample is subjected to freeze-thaw cycles, then this can cause DNA shearing.

Fortunately, alternative techniques are available that can maintain DNA integrity without the need for freezing. In this article, we’ll explore some of the methods of maintaining DNA integrity within saliva, swabs, and stool samples.

DNA Preservation using Desiccation

Desiccation, or drying, is a simple method for DNA preservation that works well for swab samples. Silica gel or similar desiccants are used to absorb moisture from the environment surrounding the DNA sample. By removing water from the sample, desiccation inhibits the activity of nucleases and other enzymes that could degrade DNA. Desiccation is particularly useful for preserving DNA in field settings where access to freezers may be limited.

Isohelix SGC Dri-Capsules are a fast and simple way of stabilizing buccal swab samples. A silica gel capsule is placed in the collection tube sitting on top of the swab shaft, maintaining DNA integrity for up to three years.[i]

Alternatively, the Rapi-Dri swab kit from Isohelix contains an easy to use buccal swab and a quick-drying pouch with a unique matrix that rapidly stabilizes samples by removing water. The pouch then acts as a sealed shipping unit for sample transport. Sample integrity can be maintained even if samples are subjected to particularly hot and humid environments. [ii]

Stabilizing DNA using Chemical Stabilization Reagents

Stabilization of DNA from Buccal Cells

Swabs are a straightforward method of collecting donor DNA.

For swabs that have been used to collect buccal cells, the collection tubes in the Isohelix BuccalFix DNA Stabilisation and lysis kit contain a buffer that has been specifically formulated to allow long term storage of swab samples at room temperature, enabling the subsequent isolation of high DNA yields and purity from the stabilized swabs.

Stabilization of DNA in Saliva Samples

Saliva samples can also be collected using swabs. To maintain DNA integrity with saliva samples, Isohelix SaliFix™ swab collectors  are recommended. The kits contain SaliFix™ SwabCatcher tubes prefilled with SaliFix™ buffer that is optimized to work with saliva swab samples

For larger volumes of saliva, e.g. 1-3ml,  GeneFix DNA/RNA collector tubes are pre-filled with a stabilization reagent that can stabilize DNA at room temperature for over five years.

If, however, you are interested in microbial DNA rather than human DNA, Isohelix has developed a new chemistry designed to maximize the collection and preservation of microbial DNA from human saliva samples. The reagent is included in the GeneFix Microbiome DNA Collector Kit, and can be used to provide a snapshot of the microbiome at the point of collection, stabilizing samples for over 12 months.

Stabilization of DNA from Stool Samples

Studies investigating individual gut microbiomes require the purification of microbial DNA from faecal samples. It is important that these samples are collected and stabilised correctly to ensure that the microbiome at the point of collection is analysed, and results are not biased by sample handling after collection.

Isohelix swabs, combined with StoolFix stabilization buffer, can be effectively used to collect and preserve microbial DNA from faecal samples. Samples are collected by brushing the stool lightly with each face of the swab and are stable at room temperature for up to two months.

Conclusion

Isohelix offers a range of DNA and RNA stabilization solutions that maintain nucleic acid integrity at room temperature for buccal cells, saliva, and stool samples. All stabilization products are optimised for use with Isohelix DNA extraction kits. 

[i] Dri-Capsule_3_Year_Study.pdf (isohelix.com)

[ii] RapiDri-Genotyping-Heat-App-Note-ver-4-020721.pdf (isohelix.com)

High molecular weight (HMW) DNA is important for applications that require the use of very long DNA molecules, such as long read sequencing. Long-read sequencing enables the detection of variants that are often difficult to resolve with short reads, such as structural variants, complex rearrangements, and variants in highly repetitive regions.

In order to fully take advantage of long-read sequencing, high quality, high molecular weight (HMW) input DNA is required. Traditional DNA extraction methods often result in fragmented DNA samples. If these shorter fragments are not removed, the average length of sequencing reads, and subsequent effectiveness of long-read analyses are reduced.

To address this requirement, Isohelix has developed the Mag-Filter HMW Clean Up Kit, which employs magnetic beads alongside specialized size selection chemistry to isolate high molecular weight DNA fragments from extracted DNA samples, while removing smaller DNA fragments and other contaminants from samples.

Samples prepared using the Mag-Filter HMW Clean Up Kit are perfect for NGS and long-read sequencing applications.

HMW DNA Cleanup and Size Selection

The Mag-Filter HMW kit removes low molecular weight DNA while safeguarding high molecular weight DNA integrity. High purity HWM DNA is achieved through DNA sample cleanup and size selection.

HMW DNA from saliva and buccal swab samples

The Mag-Filter HMW kit is optimized for use with extracted saliva samples but can be used on DNA from a variety of sample sources, such as buccal swabs. It can also be used to increase sample purity, allowing the use of valuable samples with initially low purity that might otherwise fail quality control checks.

Simple to Use HMW DNA Preparation

The kit is simple to use and can cleanup samples for library prep in less than one hour. All kit components are non-hazardous, and gel separation is not required. The kit protocol can also be easily adjusted to accommodate varying sample volumes; the protocol is fully scalable, from small manually processed sample batches up to plate-based high throughput automation.

Introduction

With advanced genomic analysis applications becoming routine in many labs, the need for high purity, high quality DNA and RNA samples collected using non-invasive sampling methods, such as saliva, is becoming increasingly important.

Nucleic acid extraction from saliva can be challenging due to the presence of contaminants and inhibitors  that can interfere with downstream applications. However, in recent years significant improvements have been made with devices to collect, store, and process saliva to extract DNA and RNA. Good yields of high-quality nucleic acids can now be extracted for use in sensitive downstream applications.

Variations in sample collection and handling can dramatically affect data quality. This article explains how to get the best from your saliva samples by maximizing the purity and yields of DNA and RNA with information on:

• Preventing sample contamination
• Saliva collection from donors
• Nucleic acid stabilization
• Safely shipping saliva samples
• Choosing your DNA/RNA isolation kit
• Isolating high molecular weight (HMW) DNA from saliva   
• Cleaning up your DNA after an initial extraction

Preventing sample contamination

The main sources of human DNA in saliva are epithelial cells and leukocytes. Saliva samples may also include bacterial DNA, allowing for DNA extraction and analysis of the oral microbiome. To prevent contamination from external nucleic acids and other factors that may interfere with the purity of samples, it is important to ensure that donors do not eat, drink, smoke, or chew gum for at least 30 minutes before providing a sample.

Saliva collection from donors

For collection by donors at home without medical supervision, kits such as the GeneFix^™ Saliva DNA/RNA range include a simple to attach funnel that makes it easy to collect saliva in a tube prefilled with stabilization reagent for transport and storage of the sample. 

Traditionally, collection devices were designed to collect 1ml or 2ml saliva, but newer devices such as the GFX-03, can collect up to 3ml if higher yields (300μg+) are required, e.g., for biobanking. For maximum yields, donors must ensure that they don’t include bubbles when measuring the liquid level in the tube as this can result in overfilling, or not collecting enough saliva.

Some donors, e.g., children or elderly patients, may find it difficult to produce saliva. If this is the case, saliva production can be stimulated by gently rubbing the cheeks or using a kit such as the SaliFix^™ Saliva Swab DNA Collection kit which contains absorbent swabs for easy collection of smaller volumes of saliva.

Nucleic acid stabilization

One of the key benefits of using saliva as a source of nucleic acids is that samples can be collected remotely and mailed for analysis. Stabilization reagents protect sample integrity by stabilizing DNA and RNA and preventing microbial growth. GeneFix saliva collection kit tubes are prefilled with a non-hazardous guanidium-free stabilization reagent that stabilizes DNA at room temperature for 5 years and RNA for 2 months. Samples can be shipped and stored at room temperature, removing the costs and logistical challenges associated with cold chain storage and transport. For longer term storage GeneFix kits are also suitable for freezing at temperatures down to -80°C.

Safely shipping saliva samples

Shipping packaging and collection tubes must be manufactured from robust materials that can withstand the rigors of the mailing process to ensure your saliva samples arrive at the lab intact. Isohelix sample collection tubes are pressure tested to 95kPa, and Isohelix^™ TPS-50 transport packs are pressure-tested and adhere to UN3373 packaging standards – including secure sealing strips and absorbent material that retains samples leaking from the primary tube within further layers of packaging, preventing sample cross-contamination.

Choosing your DNA/RNA Isolation Kit

Precipitation methods of DNA isolation such as the GeneFix^™ Saliva-Prep Kit, enable the rapid and efficient extraction of high yields of high molecular weight DNA and can be easily scaled to accommodate different sample volumes. As these kits don’t require additional solvents, columns, or filtration, workflows are easy to automate, with fast handling times and reduced sample steps. The unique precipitation chemistry maximizes recovery of high molecular weight DNA, while minimizing co-precipitation of RNA and degraded low-weight DNA.

Alternatively, silica membrane-based spin columns, such as those included in the Xtreme (XME) and Xtreme-RNA (XMR) kits, allow the purification of very high-purity, high molecular weight DNA or RNA for demanding downstream applications such as next generation sequencing or microarrays.

Isolating high molecular weight (HMW) DNA from saliva

Gentle handling is key to preserving the integrity of your DNA samples and isolating HMW DNA for more demanding applications such as whole genome sequencing (WGS) or microarrays. To prevent shearing, use wide bore tips and pipette slowly, avoiding vortexing or shaking tubes.

Cleaning up your DNA after initial extraction

If, despite your best efforts, your extracted DNA is not of a sufficient concentration or purity for your downstream applications, a DNA clean up kit can be used. The Isohelix DNA clean up kit uses a simple 30 minute protocol to reclaim a “failed” sample, preserving your DNA yields and giving High Purity DNA with 260/280 ratios to 1.8.

Nanodrop Scan Before & After DCU CleanUp

Summary

By following the advice given in this article, high yields of high purity DNA can be extracted from saliva for use in a number of sensitive downstream applications.

GeneFix offers superior performance and high yields when compared with alternative collectors :