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.
Saliva DNA samples spiked with DNA ladder and processed using the Mag-Filter Clean-Up Kit demonstrate effective removal of small DNA fragments.
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.
GFX-01
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.
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.
GFX-03
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 :
2023 was a busy year for Isohelix™ and our customers. In this blog, we present examples of how studies reported in peer-reviewed publications used Isohelix™ products to solve their sampling challenges.
We’ve highlighted four studies where Isohelix™ technologies were used to make groundbreaking discoveries in the world of biomedical research. In each of these studies, Isohelix™ sample collection products were used to collect DNA samples which were then analyzed using highly sensitive genetic analyses such as single nucleotide polymorphism (SNP) genotyping, genome wide association studies (GWAS), and next generation sequencing (NGS) analyses.
GeneFix™ Saliva Collection Devices are used to collect samples for a large population-based study in Australia
The Hong Kong Genome Project uses GeneFix™ Saliva Collection Devices to collect saliva samples and the RapiDri Pouch kit for buccal swabs.
Isohelix™ saliva collection kits are used to collect saliva from a large cohort of children in the USA
Isohelix™ Buccal Swabs are used to swab surfaces to collect DNA from bacterial communities in subway facilities
STUDY 1 : Preliminary results from the Australian Genetics of Bipolar Disorder Study: A nation-wide cohort[i]
The Australian Genetics of Bipolar Disorder (BD) Study investigates a nation-wide cohort of adults living with bipolar disorder. The study aims to identify genetic risk factors influencing BD, medication treatment response, and adverse drug reactions (ADRs). Key goals are to characterize the patterns and costs of healthcare usage in BD and to examine the relationship between genetic risk and symptom severity. This article describes the study design and sample characteristics and summarises data collected on BD symptoms, psychiatric comorbidities, and medication use.
A significant strength of the study is its sample size; it’s the largest population-based study of adults living with BD in Australia. Study participants are based throughout Australia, with many living in rural or remote communities. Saliva samples are collected using Isohelix™GeneFix™GFX-02 2 mL Saliva Collection Devices which stabilize DNA and RNA at room temperature for up to 60 months, then mailed to QIMR Berghofer via prepaid Australia Post. Following DNA extraction, genotyping is conducted using the Illumina Global Screening Array V.2.0.
GFX-02
The study demonstrates that Australians with BD are motivated to participate in genetically focused research studies if given the opportunity and provided with simple and effective sample donation protocols. GeneFix™ Saliva Collectors are ideal for this type of project as their reagents are non-toxic, and the collectors are easy to use as they have a unique funnel design that prevents buffer spillages or flow-back.
The results highlight the high rate of comorbidities and adverse drug reactions among adults living with bipolar disorder in the general Australian population and pave the way for future genomic analyses to identify genetic variants influencing pharmacotherapy treatment response and side effects.
STUDY 2 : The Hong Kong genome project: building genome sequencing capacity and capability for advancing genomic science in Hong Kong[ii]
The Hong Kong Genome Project (HKGP) is the first large-scale genome sequencing (GS) project in the Hong Kong Special Administrative Region. The project aims to sequence 45,000-50,000 genomes in five years, providing participants with diagnoses and personalized treatments and driving the application and integration of genomic medicine into routine clinical care.
For each participant, 6 mL of blood is obtained and stored in two 3-mL EDTA-containing anticoagulation tubes. For buccal swabs, the RapiDri Pouch kit is used to collect cells from inside the cheek, and saliva samples are collected using the GeneFix™ Saliva DNA Collection and Stabilization Kit. DNA from saliva samples has been shown to be comparable to DNA from blood in many downstream applications[iii] and DNA from saliva samples collected using GeneFix™ kits is stable at room temperature for over five years, avoiding the high cost and logistical challenges of cold chain transport.
RapiDri Swab
This paper describes the establishment of a robust genome sequencing workflow for the project. The laboratory analyses approximately 350-500 samples per week. During the first 24 months of the project, 12,937 participants and their family members were recruited, and the laboratory has been adapted to facilitate the latest technologies, e.g., long read sequencing and multi-omics.
STUDY 3 : Avoidant Restrictive Food Intake Disorder Genes and Environment (ARFID-GEN): study protocol[iv]
This paper describes the Avoidant Restrictive Food Intake Disorder – Genes and Environment study (ARFID-GEN), which is designed to expand the discovery of genetic and environmental contributions to ARFID risk. The goal of the study is to deliver actionable findings that can be transformed into clinically meaningful insights. This will be achieved by advancing the understanding of the genetic architecture of ARFID relative to other eating disorders and other psychiatric, neurodevelopmental, and metabolic disorders and traits.
3,000 children and adults with ARFID from the United States are included in the study. Efficient online phenotyping of children and adults with ARFID including environmental exposures is combined with at-home saliva sampling, and samples are genotyped to analyze single nucleotide polymorphism (SNP) – based heritability, genome wide association studies, genetic correlations (rgs), and copy number variations (CNVs).
SK-2S
Eligible children are asked to complete a questionnaire on their symptoms and provide a saliva sample using Isohelix™ saliva collection kits, which are non-toxic and easy for children to use at home as they have a unique funnel design that prevents buffer spillages or flow-back. The kits are provided with return packaging for mailing to the lab for analysis.
The aim is to identify genome-wide significant loci for ARFID, informative rgs, implicated CNVs, and environmental precipitants. Ultimately, the work will hopefully yield information on critical biological pathways that may point toward drug discovery or repurposing that could aid in reversing the tenacity and lethality of these illnesses.
STUDY 4 : Investigation of Surface Bacterial Diversities and Compositions in the Global Subway Facilities[v]
This study explored surface bacterial communities in four global subway facilities using 16S rRNA gene amplicon sequencing of DNA extracted from swabs.
Isohelix™ Buccal swabs can be used to collect surface DNA from environmental sources as well as from buccal samples. Indoor surface samples for Busan City were collected twice per site using Isohelix™ DNA/RNA buccal swabs (SK-2S, Isohelix) moistened with sterile 1 mL of phosphate-buffered saline. Samples were collected for 3 min from each site in a 100 cm2 area as recommended in a previous study[vi]. The microbiome was analysed based on the 16S rRNA gene.
SK-2S
Samples collected in Korea were analysed alongside comparable samples obtained from previous studies in Boston[vii], Moscow[viii], and Mexico City[ix].
The study showed that the surface bacterial diversities and taxonomic profiling of the studied subway station surface environments were similar. Most of the predominant phylotypes were Gram-positive microorganisms that probably originated from human and outdoor sources.
Summary
The above studies are just a few of the projects that have used Isohelix™ sample collection and stabilization products to collect samples from research study participants or the environment, for genomic analysis.
Tell us about how Isohelix™ products helped your study by emailing us at info@isohelix.com!
References
[i] Lind, Penelope A., et al. “Preliminary results from the Australian Genetics of Bipolar Disorder Study: A nation-wide cohort.” Australian & New Zealand Journal of Psychiatry 57.11 (2023): 1428-1442. https://journals.sagepub.com/doi/epub/10.1177/00048674231195571
[ii] Chu, Annie TW, et al. “The Hong Kong genome project: building genome sequencing capacity and capability for advancing genomic science in Hong Kong.” J Transl Genet Genom 2023;7:196-212 https://www.oaepublish.com/articles/jtgg.2023.22
[v] Kim J, Bae S, Park S, Shukla SK, Yoo K. Investigation of Surface Bacterial Diversities and Compositions in the Global Subway Facilities. Atmosphere. 2023; 14(1):140. https://doi.org/10.3390/atmos14010140
[vi] Jansson, L.; Akel, Y.; Eriksson, R.; Lavander, M.; Hedman, J. Impact of swab material on microbial surface sampling. J. Microbiol. Methods 2020
[vii] Hsu, T.; Joice, R.; Vallarino, J.; Abu-Ali, G.; Hartmann, E.M.; Shafquat, A.; DuLong, C.; Baranowski, C.; Gevers, D.; Green, J.L. Urban transit system microbial communities differ by surface type and interaction with humans and the environment. Msystems 2016, 1, e00018-16
[viii] Klimenko, N.S.; Tyakht, A.V.; Toshchakov, S.V.; Shevchenko, M.A.; Korzhenkov, A.A.; Afshinnekoo, E.; Mason, C.E.; Alexeev, D.G. Co-occurrence patterns of bacteria within microbiome of Moscow subway. Comput. Struct. Biotechnol. J. 2020, 18, 314–322
[ix] Hernández, A.M.; Vargas-Robles, D.; Alcaraz, L.D.; Peimbert, M. Station and train surface microbiomes of Mexico City’s metro (subway/underground). Sci. Rep.2020,10, 8798
Increasing numbers of saliva samples are being collected remotely from study participants for use in genetic testing, so the need for safe, simple and easy to use sample collection, stabilization and transportation is growing rapidly.
Studies have shown that DNA samples from swabs can degrade rapidly without the correct stabilization. SaliFix™ Collectors contain 1ml of preservation reagent that is designed to maintains DNA integrity and yields whilst preventing bacterial growth long term, yet provides easy and easy device for Saliva DNA collection, stabilization, and transport.
SaliFix™ Collectors are specifically designed for collecting Saliva samples in various environment including at home, in clinic or remotely. The foam swabs effectively maximizes saliva collection, even from donors who may find it difficult to produce saliva.
The SaliFix™ kit also includes the SwabCatcher format for improved Sample Handling
The NEW SwabCatcher tube simplifies sample processing and cuts down on cross-contamination as the cap holds the swab tightly after screwing the cap onto the tube. This removed the tedious process choice of having to cut the swab tip and or manually using tweezers to remove the swab prior to processing. This makes the tube ideal for high-throughput processing using automated instruments, and further the tubes are barcoded for easy sample tracking.
SwabCatcher tubes are leakproof and fully transport tested to 95kPa to allow mailing and storage, and the reagents are non-hazardous.
Agarose gel analyzed by electrophoresis. Saliva DNA isolated from SaliFixTM collection devices using the Isohelix Saliva-Prep-2 kit (GSPN-50), shows high quality DNA with no degradation.
DNA collected using SaliFix™ collectors is of high yield and purity, and suitable for all downstream analyses.
Saliva Swab DNA Collection Kit
All-in-one saliva DNA collection, stabilization, and transport device.
Uses the NEW Swab-Catcher for easy handling and automated-processing.
Simple collection at home, in clinic. a viable alternative to blood collection.
Instant stabilization maximises DNA yields allowing RT shipping and storage in a non-hazardous format.
Optimized for extraction using Isohelix DNA extraction and other kits.
Product Information
SFX/LS/1/50 SaliFix™ saliva collection tubes prefilled with 1ml SaliFix™ buffer and individually wrapped, foam saliva swabs.
NEW SwabCatcher tubes are used alongside RapiDri DNA and RNA Swab Kits designed to simplify the swabs transition through sample processing. The Tubes are also available pre-filled with BuccaLyse DNA and RNA stabilization buffer for room temperature shipping.
No more Swab retrieval issues
Automation, HT and Manual compatible
Barcoded for traceability
CE-IVD Certified
Fully transport tested to 95kPa
Product Information
SwabCapture L/00/50 50 x SwabCatcher 5ml collection tubes and caps(empty)
RapiDri RD-01 200 x RapiDri swabs SwabCatcher with drying pouch
BuccaLyse BEK/L/05/50 50 x BuccaLyse SwabCatcher collection tubes pre-filled with 0.5ml buffer
BuccalFix BFX/L/05/50 50 x BuccalFix SwabCatcher collection tubes pre-filled with 0.5ml buffer
At Isohelix we use our extensive knowledge in product design, regulatory, safety & commercial markets to design products that make it easy for our customers to collect high quality nucleic acid samples from saliva, buccal cells, and stool samples.
Isohelix will be present at the upcoming Festival of Genomics & Biodata in London, Booth #79, from 24th to the 25th of January. Join us to explore our latest products in Sample Collection, Stabilization, and Purification technologies.
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