Assessing CLIA / CAP Certified Next Generation Sequencing Facilities

clia-ngs-lab

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:

Assessment Test Next Generation Sequencing Specification Sample Material
Accuracy Coverage and Quality or Phred Scores Known variants (SNP, indel) in targeted region
Precision Sequence replication and coverage distribution between different operators and instruments Reference with known variants
Specificity False positive rate, degree with which a false variant is identified at a specific coverage threshold Several samples with well characterized targets
Sensitivity Likelihood test detects known variant Several samples with well characterized targets
Reportable Range Intron buffer and exon region of one or more genes Target material with repeat regions, indels, allele drop outs
Reference interval Sequence variation background measurement Derived from an unaffected population, same as patient

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.

Isolation of cell free / circulating tumor DNA from plasma

tubes

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.

Kit Company Method Digestion Prep Time (min) Plasma Volume (mL) Elution (uL) DNA sizes

(bp)

NextPrep-Mag

 

Bioo Scientific Mag Beads Proteinase K (optional) 30 1 – >5 12 >50
Chemagic cfNA Chemagen Mag Beads Proteinase K 120 2 – 10 60 >100
MagMAX Cell Free DNA Kit Thermo Fisher Mag Beads Proteinase K

(optional)

40 1 – >5 15 >50
QIAamp

 

Qiagen Column Proteinase K 120 1-5 20 >70
Quick-cfDNA

 

Zymo Research Column Proteinase K 60 3- 10 35 >100

Targeted gene panels vs. whole exome sequencing

gene-panels

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.

Guides to improve your next generation sequencing project

read length, output and instrument recommendations for next generation sequencing

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.

 

6 Methods to Fragment Your DNA / RNA for Next-Gen Sequencing

The preparation of a high quality sequencing library plays an important role in next-generation sequencing (NGS). The first main step in preparing nucleic acid for NGS is fragmentation. In the next series of blog posts we will present important challenges and things to consider as you isolate nucleic acid samples and prepare your own libraries.

Next Generation Sequencing, will give you a plethora of reads, but they will be short. Illumina and Ion read lengths are currently under 600 bases. Roche 454 outputs reads at less than 1kb and PacBio less than 9kb in length. This makes sizing your input DNA or RNA important prior to library construction. There are three main ways to shorten your long nucleic acid material into something compatible for next-gen sequencing: 1) Physical, 2) Enzymatic and 3) Chemical shearing.

Physical Fragmentation

1) Acoustic shearing

2) Sonication

3) Hydrodynamic shear

Acoustic shearing and sonication are the main physical methods used to shear DNA. The Covaris® instrument (Woburn, MA) is an acoustic device for breaking DNA into 100-5kb bp. Covaris also manufactures tubes (gTubes) which will process samples in the 6-20 kb for Mate-Pair libraries. The Bioruptor® (Denville, NJ) is a sonication device utilized for shearing chromatin, DNA and disrupting tissues. Small volumes of DNA can be sheared to 150-1kb in length. Hydroshear from Digilab (Marlborough, MA) utilizes hydrodynamic forces to shear DNA.  Nebulizers (Life Tech, Grand Island, NY) can also be used to atomize liquid using compressed air, shearing DNA into 100-3kb fragments in seconds. While nebulization is low cost and doesn’t require the purchase of an instrument, it is not recommended if you have limited starting material. You can lose up to 30% of your DNA with a nebulizer. The other sonication and acoustic shearing devices described above are better designed for smaller volumes and retain the entire amount of your DNA more efficiently.

Enzymatic Methods

4) DNase I or other restriction endonuclease, non-specific nuclease

5) Transposase

Enzymatic methods to shear DNA into small pieces include DNAse I, a combination of maltose binding protein (MBP)-T7 Endo I and a non-specific nuclease Vibrio vulnificus (Vvn), NEB’s (Ipswich, MA) Fragmentase and Nextera tagmentation technology (Illumina, San Diego, CA). The combination of non-specific nuclease and T7 Endo synergistically work to produce non-specific nicks and counter nicks, generating fragments that disassociate 8 nucleotides or less from the nick site. Tagmentation uses a transposase to simultaneously fragment and insert adapters onto dsDNA. Generally enzymatic fragmentation has shown to be consistent, but worse when compared to physical shear methods when it comes to bias and detecting insertions and deletions (indels) (Knierim et al., 2011). Depending on your specific application, de novo genome sequencing vs. small genome re-sequencing, biases associated with enzymatic fragmentation may not be as important.

RNAse III is an endonuclease that cleaves RNA into small fragments with 5’phosphate and 3’hydroxyl groups. While these end groups are needed for RNA ligation, making the assay convenient, RNAse III cleavage does have sequence preference which makes the cleavage biased. Heat / chemical methods described below, while they leave 3’phosphate and 5’hydroxyl ends, show less sequence bias and are generally preferred methods in library preparation.

Chemical Fragmentation    

6) Heat and divalent metal cation

Chemical shear is typically reserved for the breakup of long RNA fragments. This is typically performed through the heat digestion of RNA with a divalent metal cation (magnesium or zinc). The length of your RNA (115 bp – 350 nt) can be adjusted by increasing or decreasing the time of incubation.

The size of your DNA or RNA insert is a key factor for library construction and sequencing. You’ll need to choose an instrument and read length that is compatible with your insert length. You can choose this by entering project parameters in the Shop by Project page and filtering according to read length (estimated insert length). If you’re not sure, we can help. Send us a request through our consultation form .

Reference:

Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

Ellen Knierim, Barbara Lucke, Jana Marie Schwarz, Markus Schuelke, Dominik Seelow

 

 

World’s Leading NGS Matching Engine Just Got Better

Every week thousands of researchers from around the world rely on Genohub’s free next-generation sequencing matching engine to explore sequencing solutions that match their project requirements. This typically involves finding out the right amount of capacity (e.g. number of sequencing lanes) on different sequencing platforms to meet a coverage or read count requirement for various applications such as DNA-Seq, RNA-Seq, Exome, Amplicon-Seq, etc.

We are very excited to release a brand new version of our NGS matching engine, that packs a lot of major improvements. Here are a few highlights:

Redesigned Interface

We have completely redesigned the interface to make it even faster and easier to find matching services. It’s now also easier to quickly send a request to get confirmed quotes or to get help from our scientists

genohub-ngs-search-interface

Detailed Quote View

You can now view detailed quotes right from the results. This makes it a lot easier to evaluate different options based on additional service details.

genohub-inline-quote

Instrument and Library Preparation Kit Filters

You can now filter the results by instrument or library preparation kit. This helps with situations where for whatever reason (e.g. consistency with a previous sequencing run) you’d like to stick with a particular sequencing instrument.

genohub-instrument-filtergenohub-library-prep-kit-filter

Expanded Range of Services and Lower Prices

We’ve been working very hard with our network of partnering service providers to expand the range of sequencing services. Here are just a few examples:

  • New instruments such as 10X Genomics, HiSeq X, HiSeq 3000/4000, NextSeq 500
  • New applications such as Ribo-Seq, targeted amplicon, mtDNA, HLA and TCR-repertoire sequencing
  • Gene panels such as Qiagen’s amplicon panels, Agilent and Nimblegen’s Target Hybrid capture panels and IDT’s xGEN Lockdown panels.

Whether you are just exploring options for a future project, looking to get a few quotes for a grant application, or have an immediate sequencing project with samples ready to ship, there’s no better way to find and order the right NGS services. As always, we’d love to hear your feedback on what you like, and more importantly what else you’d like to see improved. Leave a comment here or email us at support@genohub.com.

To use the service visit Genohub.com.

Considerations for Sequencing microRNA

microRNA sequencing

We’ve put together a new small RNA (microRNA) sequencing guide describing considerations all new users should make before undertaking a small RNA sequencing project. One of the first considerations is determining the number of reads you need. This usually depends on whether you’re interested in differential small RNA expression or if you’re trying to discover new microRNAs. Once you know the number of reads you need per sample, consider the following factors before and after library preparation:

  1. Should you start with total RNA or isolated small RNA?
  2. How much material should you start with?
  3. What’s the minimum quality of total RNA acceptable for microRNA library preparation and sequencing?
  4. How will small RNA ligation bias affect my results?
  5. How can I minimize adapter dimers to improve read mapping and general usability of my sequencing reads?
  6. How many samples can I multiplex or pool together in a single sequencing lane?
  7. What sequencing read length should I choose for microRNA or small RNA sequencing studies?

The guide also includes recommendations for getting accurate per sample pricing and turnaround times.

Small RNAs play a big role in regulating the translation of target RNAs through RNA to RNA interactions and have been shown to offer potential as biomarkers in diagnostic applications. Sequencing promises to be a useful tool in unraveling the roles of these short non-coding RNAs. We look forward to working with you on your next microRNA project.