Illumina sequencing follows three very simple steps:

1. Libraries are prepared from DNA or RNA samples
2. Single molecular DNA templates are bridge amplified to form clonal clusters inside a flow cell
3. Clusters are sequenced by massive parallel synthesis

Template molecules are immobilized on a flow cell surface and amplified by isothermal bridge amplification to create  individual dense clonal clusters containing ~2,000 molecules each (see figure above).

The exact density of these clusters can influence:

1. Run quality
2. Reads passing filter
3. Q30 scores
4. Total number of reads

This makes proper loading of an Illumina flow cell crucial to the success of a sequencing run.

In a recent guide, we review recommended loading concentrations and cluster densities for each Illumina instrument. See a summary in Table 1. below:

While this table includes recommendations for standard library applications where libraries are sufficiently diverse, researchers shouldn’t follow these recommendation for libraries that have poor diversity. Sequence diversity refers to the balance of nucleotides (A, T, G, C) at each position of a template library. Applications where you should load a library at a concentration below Illumina’s standard recommendations include:

1. Any amplicon based library where primers are included in the read insert
2. GBS or RAD-seq libraries that start with a similar restriction site
3. 16S or 18S libraries that start with the same primer or variable domain sequence
4. MeDIP or other low diversity targeting approach

If you’re working with a non-standard library preparation application or one where libraries have poor sequence diversity, submit a request here: genohub.com/ngs and a scientist will recommend flow cell loading concentrations.

According to the Centers for Medicare and Medicaid Services (CMS), Clinical Laboratory Improvement Amendment (CLIA) registration is required for entities that perform a single test on, “materials derived from the human body for the purpose of providing information for the diagnosis, prevention or treatment of any disease or impairment of, or the assessment of the health of, human beings”.

To date, only two next generation sequencing (NGS) instruments/tests have been approved or cleared by the FDA. All other NGS based tests are developed in house as laboratory developed tests (LDTs), and are regulated under CLIA. CLIA regulations are required to certify the validity of a test. Validity is established by measuring:

1. Accuracy
2. Precision
3. Analytical sensitivity and specificity
4. Reportable reference range or interval

For next generation sequencing tests this means several sequencing based metrics are required:

In addition to CLIA, the College of American Pathologists (CAP) has several specific guidelines for NGS labs. These include consideration for validated sample extraction, library preparation, barcoding, pooling and target enrichment. Each protocol has specific quality metrics associated with it. In addition to the wet lab, bioinformatics pipelines must be validated and tested for how precise and sensitive variants are called.

Clinical regulation of NGS based tests are undergoing rapid change as new NGS tests enter the clinic, and older ones are improved. As these changes happen, both CAP and CLIA requirements for NGS are updated on a yearly basis.

The most common NGS based assays or tests performed in a CLIA/CAP setting today include:

1. Exome sequencing
2. NGS gene panel sequencing
3. Whole genome sequencing
4. Cell free DNA sequencing
5. Metagenomic sequencing

Genohub has existing relationships with 7 service providers offering nucleic acid extraction, library preparation, sequencing and data analysis under CLIA and CAP. To obtain NGS services under CLIA/CAP accreditation, submit a request here: https://genohub.com/ngs.

Identification of biomarkers that indicate presence of disease are highly sought after. Non-invasive methods to measure those biomarkers are even more valuable. By extracting and measuring cell-free DNA, scientists have satisfy both.

Cell free DNA are degraded fragments released in plasma. Elevated levels of cfDNA are found in cancer states, making assessment of somatic genomic alterations from tumors possible using sequencing. Cell free fetal DNA (cffDNA) can be found as early as 7 weeks gestation, and analysis of cffDNA is already being used in non-invasive prenatal diagnostics. Cell free DNA (cfDNA) in blood was first described by Mandel and Metais in 1948 [1] but only recently has been identified as having utility for prenatal testing and disease diagnostics and monitoring.

Unlike mutations that are passed from a parent to child and are in every cell of your body, somatic mutations form during a person’s life. These somatic mutations are present in tumor cell DNA and are an excellent biomarker if they can be measured and monitored.

Acquiring tumor DNA often requires a biopsy, a potentially risky and invasive procedure. In many cases presence of a tumor or the ability to biopsy is not even an option for patient. During tumor turnover and progression, apoptotic and necrotic cells release small pieces of their DNA (cfDNA) into the bloodstream. The amount of cfDNA in the blood steam is influenced by clearance and filtering of the blood and lymphatic circulation.

Detecting cfDNA in plasma is called a ‘liquid biopsy’ and is already a popular method for obtaining clinical samples for prenatal testing, disease diagnostics and monitoring. One of the challenges of liquid biopsies, are standardization of the isolation procedure and maintaining  uniform specificity and sensitivity. Extraction of cfDNA can be carried out using magnetic beads or silica matrices along with chaotrophic salts, such as guanidine thiocyanate. While several commercial approaches (Table 1) exist, none have undergone rigorous large patient scale studies. Once more information is known, universal standardization should allow greater clinical utility.

Commercial kits for extraction of cfDNA need to be designed to extract uniform DNA copies from varying biopsy volumes. Scalability and adaptability for cell free fetal and ctDNA are important considerations. Below we highlight current kits available in the market. In a future blog post we’ll discuss isolation and sequencing standardizations required for broader use of cfDNA liquid biopsy.

Table 1.

One frequent question we hear on Genohub is, ‘should I make a custom panel for this gene set, or not bother and do whole exome sequencing?’. While whole genome sequencing approaches can capture all possible mutations, whole exome or targeted gene panel sequencing are cost-effective approaches for capturing phenotype altering mutations. We go into the advantages of WGS vs. WES in an earlier blog post. A remaining question however is, among targeting approaches, which is best. We attempt to address this here:

Advantages of targeting all exons – whole exome sequencing (WES)

If your study is discovery based, in other words you don’t know what genes you need to target, WES is the obvious choice.

• Better for discovery based applications where you’re not sure what genes you should be targeting.
• Exome panels are commercially available, they don’t need to be customized or designed.
• Exome sequencing services are fairly standard, costs range between $550-800 for 100-150x mean on target coverage.

Advantages of targeted gene panels (amplicon-seq or targeted hybridization methods)

Targeted gene panels are ideal for analyzing specific mutations or genes that have suspected associations with disease.

• Focusing on individual genes or gene regions allows you to sequence at a much higher depth than exome-seq, e.g. 2,000-10,000x as opposed to 200x which is typical with exome-seq.
• High depth sequencing enables the identification of rare variants
• Can be customized for different samples types, e.g. FFPE, cf/ctDNA, degraded samples.
• Lower input amounts can be used with targeted gene panels (1 ng vs. 100 ng with whole exome sequencing).
• Gene panels can be customized to only include genomic regions of interest. Why sequence everything when you don’t need that extra information?
• Panels can be easily designed for non-human species. Designing a non-human exome is much more laborious.
• Gene panel workflows are a lot simpler and time to results is often as little as 1-2 days.
• You can process thousands of samples on a single sequencing run. Targeted gene panels can be run at a higher throughput and are often more cost-effective than whole exome sequencing.

By focusing on genes likely to be involved with disease, you can reduce expense and focus sequencing resources on your targeted region. However, if you only have a few samples that you need to sequence at a low depth of coverage, consider whether it’s worth designing a panel vs. performing whole exome sequencing using an existing commercial panel.

If you’re interested in designing a custom gene panel or already have an existing panel you’d like to sequence, submit a request describing your project or view several of the existing commercially available panels here.

If you’re new to next generation sequencing or if you’re simply looking for tips to improve your next project, we recommend you take some time to look at the guides available on Genohub. As researchers order sequencing services it’s completely normal for there to be numerous questions related to nucleic acid extraction, library prep and best practices for loading a sequencing instrument. Over the years, we’ve curated these questions and published guides to help those embarking on their next NGS project. Topics covered include: library prep applications, batch effects, optimal cluster densities, read lengths and instrument output.

Next generation sequencing is a tool that can be applied to answer any number of questions related to the genome, transcriptome or epigenome. Regardless of the organism being sequenced or the library method used to prepare nucleic acid from that organism, the fundamentals of how a sequencing platform works, is similar across all samples. There are currently four main sequencing platforms that researchers regularly use. These include Illumina, Ion, PacBio and Oxford Nanopore. The guides below tend to be Illumina focused because that’s the platform most people are currently using today. Despite that, we review the read throughput of each available instrument and discuss hybrid methodologies where short and long reads are combined from two different instruments to improve assemblies.

Guides for sequencing

1. Designing a sequencing project
2. Recommended coverage by library preparation application
3. Comparison of instrument read lengths and read outputs

Guides for preparing your samples

1. Best practices for shipping tissue and nucleic acid 
2. Library preparation kits and tips

Guides by application

1. Transcriptome and mRNA-Seq
2. Genome sequencing and re-sequencing
3. Exome
4. Metagenomics 
5. Small RNA (microRNA)
6. WGS vs. WES

Tips and considerations for commonly used sequencing instruments

1. HiSeq X
2. HiSeq 3000/4000
3. Nextseq and low diversity

These are evolving guides, meaning our goal is to continuously improve them. If you have any feedback or would like to contribute please send us a message. We hope these guides will be helpful in designing your next NGS run. If you have technical questions related to an upcoming NGS project, feel free to submit them on our consultation page.

Whenever you order a scientific related service or outsource research, there is a degree of trust that is naturally built into the relationship. As a researcher you expect the service provider to take care of your precious samples and complete a service to your expectations. The service provider expects to work with researchers who have complied with sample requirements and will be reasonable when it comes to unexpected situations. This is especially the case when it comes to the relationship between a researcher and next generation sequencing service provider. Having worked with both researcher and sequencing service providers on hundreds of real projects, we’ve developed a standard quality policy for all services that happen through Genohub.com. We’ve developed this policy to ensure a trusted and efficient environment for clients and providers to work together. By setting expectations for both the researcher and provider we’re improving the way sequencing services are being performed.