Data Analysis for NGS by Ion Torrent – Part One – How Did the Run Perform?

Here comes the fun part.  It’s taken a day for library prep, an overnight run for the clonal amplification; the next day includes loading the chip with the ISPs and then running the chip on the sequencer.  After the chip has run on the sequencer, the data is pushed from the sequencer (the PGM) to the server connected to the sequencer.  This aspect of NGS surprised me – the size of the files is amazing – for one 316 chip, the file that includes all of the raw data averages about 100GB.  To deal with this amount of data, the server attached to the sequencer is 12TB, and even still we have to have a procedure to deal with removing files off that sequencer to keep space for future runs.

Anyway, the raw data is pushed to the server and the data analysis begins.  The Torrent Suite Software first analyzes the ISP info, as shown in the graphic below.  It gives a “heat map” of the chip (the football shape) in which red means the wells in those areas were full with ISPs.  Yellow means there are fewer ISPs and blue means there are none.  So, you can see below, there is a small area of blue within the football shape – this area did not have any ISPs in it.  92% of the wells on this chip were filled, however, which is about the max a chip can be loaded.

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These ISPs are then broken down into categories.  First, how many of the wells had ISPs in them – here, 92.5% of the 6,337,389 wells contained ISPs.  Of those ISPs, 99.8% of them have product on them that can be sequenced (Live ISPs).  Of those Live ISPs, 0.4% of them contain control Test Fragments and 99.6% of them contain actual patient sample library amplicons.  The Test Fragments are spiked in prior to sequencing and act as a control to evaluate how the sequencing run performed.  Lastly, the ISPs that contain patient sample library amplicons are analyzed.  Those ISPs that contain more than one amplicon (say it has an amplicon of EGFR Exon 19 and another specimen’s amplicon of KRAS Exon 2) give mixed signals and cannot be analyzed, so they are thrown out of the data analysis and into a bin called “polyclonal”.  Low quality ISPs are also thrown out – anything that did not pass the thresholds for quality.  And lastly, ISPs that only contain adapter dimers are thrown out.  For a run of AmpliSeq Cancer Hotspot Panel v2 specimens, most of which come from FFPE specimens that are low quality to start with, a run that contains over 50% Final Library ISPs is actually a very good run, interestingly enough.  The 316v2 chips are rated to sequence 1 million reads (each ISP yields one read), and on this example run, over 3 million reads were sequenced, so this is a successful run.

After the ISPs are analyzed and the high quality ones are kept, the analysis goes on.  The Torrent Suite software then calls the bases based on the raw flow data.  These bases are then aligned to a reference, in our case hg19, a commonly used human genome reference.  Quality scores are assigned at this point.  A Phred-based quality score is used for NGS, shown in the table below.

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Lastly, the reads are put into bins based on the barcode that was used for each patient specimen – remember the small part of the adapter that was added in library prep so that the specimens could be mixed together?  The software reads that adapter sequence then assigns each read based on those sequences.  The specimens should all have approximately the same number of reads since they were normalized to the same concentration at the end of library prep, but there may be some variability due to specimen quality, as you can see below.

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In next quarter’s post, we will dive into the individual specimen results!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

Next Generation Sequencing – Ion Torrent Semiconductor Sequencing

We’ve finally made it to the sequencing step of the NGS workflow. This post we will discuss the technology and process behind the Ion Torrent sequencing step. Next time, we will review the Illumina sequencing process.

When we left off, the final product of the clonal amplification had been prepared – Ion Sphere Particles (ISPs) covered in single stranded amplicons (hopefully all of the same amplicon). Next, control Ion Sphere Particles are added to the mix, along with sequencing primer, which is complimentary to one of the adapter sequences added back in library preparation. The primer is annealed to each of the amplicons on every ISP. This mixture of control ISPs and specimen ISPs is then loaded onto the chip. The size of the chip is determined by the number of bases needing to be sequenced. There are three different types of chips for the Personal Genome Machine (PGM) – 314, 316, 318 – and five different types for their GeneStudio S5 system (510, 520, 530, 540, 550), offering enough coverage for a single sample of a hotspot panel, all the way up to enough coverage for a specimen of exome sequencing. Each of the chips contains a top layer covered in tiny wells. Each well is just large enough to fit a single ISP. The ISP solution is loaded onto the chip, then flowed over it by centrifuging it in different directions, in order to attempt to get as many ISPs into wells as possible. The chip is then ready for sequencing.

Each well of the chip can be thought as of the smallest pH meter in the world. So before sequencing can be started, the instrument must be prepped (initialized) so that all of the reagents added to the chip are in the correct pH range. On the PGM, this takes approximately an hour and requires some hands-on steps and high quality 18MΩ water. On the GeneStudio S5, the reagents are added and the initialization is begun and, as long as everything works correctly, doesn’t require any other hands on time.

After the initialization is complete, the chip is loaded onto the instrument. The sequencing run is started and runs according to the plan prepared before the run. Thermo Fisher’s Ion Torrent uses semiconductor sequencing technology. Nucleotides are flowed over the chip one at a time. If the nucleotide is incorporated, a hydrogen ion is released. This release of hydrogen decreases the pH of the liquid surrounding the ISP. This pH change is then detected by the sensing layer beneath the well, where it is converted to a voltage change and is picked up by the software and recorded as that nucleotide. Let’s say two nucleotides in a row are incorporated (two G’s complementary to two C’s) – double the hydrogen is released, which results in double the signal, so the software will record two G’s in a row. The benefit of this type of technology is that it is fast – it only takes 15 seconds for each nucleotide flow, so a 200bp fragment can be sequenced in less than 3 hours.

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Image courtesy of http://www.genomics.cn/en/

 

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

CMS’s National Coverage Determination for Next Generation Sequencing (NGS) – What Does This Mean for the Future of NGS Testing for Molecular Oncology?

I thought I’d take a break from the next generation sequencing (NGS) wet bench description this month to review news occurring in the world of reimbursement of testing of cancer specimens with next generation sequencing.  As a tech, I don’t deal with the nitty gritty of insurance reimbursement of our tests on a day to day basis, but this one caught my eye as it would have had a real impact on the NGS testing in our lab.  On November 30th, 2017, a proposal was released by the Centers for Medicare & Medicaid Services (CMS) to review the national coverage analysis tracking sheet for NGS for Medicare beneficiaries with advanced cancer.  In the original wording of the proposal, one thing it stated was that CMS should only reimburse NGS testing for advanced cancers when the testing was done with an FDA approved assay.  This caught me, as well as many others in the molecular community, by surprise.  The reason?  Currently, there are only a few FDA approved assays on the market; much of the testing occurring right now for oncology assays by NGS are lab-developed tests (LDTs), including the ones that we run in our lab.  Under the proposal’s language, these types of assays would not be reimbursed for Medicare patients (and where CMS reimburses, the major insurance companies follow), making it very difficult for us to continue the testing that we perform.

The process for a proposal such as this one includes posting the proposal, then allowing a period for public comments about the proposal.  Six weeks were given for people to post their comments online, during which, 315 comments were left.  These comments included praise to CMS for recognizing that NGS testing is increasingly useful for precision medicine, but also stressed the limitations of only allowing FDS approved assays to be used.  Some comments pointed out how clinicians and pathologists work together in the institutions performing the NGS assays in a way that would be impossible if forced to use an assay from an outside institution.  They also indicated how difficult it would be for all NGS testing to be performed by the very small number of FDA approved assays and how it is almost impossible for small academic institution labs to get FDA approval for assays due to the amount of money and time the approval process takes.

On March 16, 2018, the final decision memo was released with altered wording compared to the original and can be found here https://www.cms.gov/medicare-coverage-database/details/nca-decision-memo.aspx?NCAId=290 and is also shown below:

“A.  Coverage

The Centers for Medicare & Medicaid Services (CMS) has determined that Next Generation Sequencing (NGS) as a diagnostic laboratory test is reasonable and necessary and covered nationally, when performed in a CLIA-certified laboratory, when ordered by a treating physician and when all of the following requirements are met:

  1. Patient has:
    1. either recurrent, relapsed, refractory, metastatic, or advanced stages III or IV cancer; and
    2. either not been previously tested using the same NGS test for the same primary diagnosis of cancer or repeat testing using the same NGS test only when a new primary cancer diagnosis is made by the treating physician; and
    3. decided to seek further cancer treatment (e.g., therapeutic chemotherapy).
  2. The diagnostic laboratory test using NGS must have:
    1. FDA approval or clearance as a companion in vitro diagnostic; and
    2. an FDA approved or cleared indication for use in that patient’s cancer; and
    3. results provided to the treating physician for management of the patient using a report template to specify treatment options.
  3. The diagnostic laboratory test using NGS must have:
    1. FDA approval or clearance as a companion in vitro diagnostic; and
    2. an FDA approved or cleared indication for use in that patient’s cancer; and
    3. results provided to the treating physician for management of the patient using a report template to specify treatment options.
    4. Other

Medicare Administrative Contractors (MACs) may determine coverage of other Next Generation Sequencing (NGS) as a diagnostic laboratory test for patients with cancer only when the test is performed in a CLIA-certified laboratory, ordered by a treating physician and the patient has:

  1. either recurrent, relapsed, refractory, metastatic, or advanced stages III or IV cancer; and
  2. either not been previously tested using the same NGS test for the same primary diagnosis of cancer or repeat testing using the same NGS test only when a new primary cancer diagnosis is made by the treating physician; and
  3. decided to seek further cancer treatment (e.g., therapeutic chemotherapy).

See Appendix D for the NCD manual language.”

 

In part B, it addresses those assays that are not FDA approved, but are run in a CLIA-certified laboratory.  This part was added in the final decision and makes it possible for non-FDA approved assays run in CLIA-certified laboratories to be reimbursed, dependent upon the local MACs.  While this is a huge improvement over the previous, there are still questions regarding some of the wording and we will have to see how this affects testing for our patients.  For example, in 1b, where it mentions repeat testing – some patients have multiple mutations that are followed over time for hematological malignancies – will this be considered repeat testing? It will remain to be seen.  Needless to say, I am happy to be able to continue doing my job.

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

When Rapid Blood Culture Identification Results Don’t Correlate, Part 2: Contamination

All laboratories are prone to contamination events. Blood products, analyzers, reagents, media, etc. all have the potential to be contaminated. If you are a molecular microbiologist, then you have to worry about not only bacterial, but also nucleic acid contamination. 

The Issue

The topic of my blog last month focused on discrepant results between blood culture and PCR. Traditional blood culture workflow involves correlating the Gram stain result to what grows in culture. Nowadays, many laboratories are also performing PCR on positive blood cultures. Because we know PCR is more sensitive, it may be easy for some to justify discrepancies. Let’s image that gram positive cocci in clusters were observed in the Gram stain, the PCR detected Staphylococcus and Enterococcus DNA, but only S. aureus grew in the culture. Where did the Enterococcus come from and where did it go? It was not observed in the Gram stain and it didn’t grow in cultures, so was it “real”? Possibly. It could be a contaminant or it could be real, just present in low numbers. It’s difficult to say without having to invest more effort.

When this type of situation occurs in my laboratory, three things happen. First, we review the data. For example, if the Gram stain is discrepant, then we review the Gram stain or perform an acridine orange stain (in the case of positive PCR, but negative culture). If it’s the PCR, then we would make sure that a result entry error did not occur, etc. Second, we add the comment, “clinical correlation needed”. We have found little value in going back to the blood culture bottle and trying to recover the missing organism because in most cases when we look hard enough, using selective agar and other strategies, we do find the organism from the PCR results buried among overgrowth. Therefore, our approach is to let the clinician know that they must use other clinical data to aid in their diagnosis. Third, we document all discrepant blood culture PCR results; which includes an automatic notification to the doctoral director.

Next, let’s imagine that two more blood cultures (from different patients) become positive all within a relatively short period of time from the first discrepant result noted above. gram negative bacilli are observed in one culture and the other displays gram positive bacilli. PCR detects Enterococcus DNA in both cases. What are the odds of that happening? Not good. Something strange is going on!

The Solution

A contamination investigation needs to immediately occur. The two likely sources of contamination are 1) the PCR assay or 2) the blood culture bottles. To determine whether the issue is due to amplicon or target contamination of the PCR assay, we need to identify which instruments reported the Enterococcus. Was it a single instrument or were different instruments involved? Our laboratory performs routine “swipe” tests of the environment as part of our quality control, which allows us to monitor contamination. Swipe tests may also be performed 1) after a known contamination event (i.e., spill due to cracked or leaky product) to ensure that decontamination was properly carried out, 2) to investigate increased positivity rates, or 3) follow up on unusual results, such as the scenario outlined above.

PCR may be performed on a random sampling of uninoculated bottles to determine whether the issue is due to contamination of the blood culture media. If the contamination is high density, this may be useful; however if it is low density, then all bottles you test may still be negative. If the contamination is due to bacterial DNA, then Gram stain or culture will not be useful, hence the need for PCR. It is important to note that the presence non-viable organisms and/or nucleic acids (at levels that can be detected by PCR) is a known limitation noted in the package insert of some blood culture media and PCR manufacturers. If contamination is suspected, then immediately file a report with the manufacturer. Be sure to document lot numbers and expiration dates so that they may alert other customers.
The Conclusion

Human error contributes to the majority of discordant laboratory results. However, errors in interpretation and result entry/clerical errors are only part of the problem. Contamination events only complicate matters. If the test volume is significant, then the number of discordant results should be quickly realized, especially if there truly is a contamination issue. It is important to have a process in place to help reconcile contamination events as quickly as possible as they have the potential to majorly impact operations and patient care.

 

References

  1. https://labmedicineblog.com/2018/02/20/when-rapid-blood-culture-identification-results-dont-correlate-part-1-clinical-correlation-needed/

 

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-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Template Preparation, Clonal Amplification, and Cluster Generation – Oh My! – Step Two in an NGS Setup

Hello again – let’s continue our discussion of Next Generation, or Massively Parallel, Sequencing and how it is performed.  Over the last two blogs we have seen why NGS is being used in a Molecular Diagnostics Lab and how library preparation is executed.  Specifically, we reviewed how Ion Torrent and MiSeq libraries may be prepared for DNA amplicon sequencing.  The final product of this work is a collection of amplicons that have been amplified, barcoded, tagged with the appropriate platform adapters and purified.  These are what compose a specimen’s “library.”

The next step in NGS preparation is template preparation.  The main goal of this step is to create multiple copies of the same amplicon in close proximity so that when it is sequenced, it creates a strong enough signal to be detected.  This occurs for each amplicon in the specimen’s library.  Again, this technique is platform specific, so each has a different way to achieve this goal.

Ion Torrent “Template Preparation by Emulsion PCR” or “Clonal Amplification”

In the Ion Torrent method of template preparation, the multiple copies are created on an Ion Sphere Particle or ISP.  This looks like a bead with primers all over the surface of it.  Eventually this ISP will be deposited in a well on a chip and be sequenced.  In order for this ISP to create enough of a signal to be detected by the instrument, it must have many copies of the fragment all over the surface of the ISP.

At the beginning of the clonal amplification step, a specific concentration of combined libraries is added to the instrument, along with all the components of a standard PCR (buffer, dNTPs, polymerase) with the addition of the Ion Sphere Particles, which provide the primer, and oil.  The primers on the ISP are complementary to one of the adapters added during library preparation so that only the universal primer is necessary on the ISPs, instead of each individual gene-specific primer.  Through a series of steps, ideally, what is produced is a droplet of oil containing one ISP, one sample’s amplicon, and the components of the master mix.  This, along with millions of other ISPs in droplets of oil, will undergo cycles of PCR, with the primers on the ISP priming the specimen’s amplicon.  These amplicons will replicate all over the ISP, and as a final step, NaOH will be added to separate the strands.  The strands that are not anchored to the ISP by the universal primer will be lost, leaving each ISP single stranded and ready for priming in the sequencing step.

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One thing to consider is the concentration of the combined libraries that are added at the beginning of the template preparation.  If the concentration is too low, obviously not enough amplicons will be amplified on the ISPs, and the end result will be not enough data.  Conversely, if the concentration is too high, there is a possibility of more than one sample amplicon ending up in the droplet of oil.  In the end, more than one fragment gets amplified on the ISP.  This ISP is called “polyclonal” and the data from it will get thrown out.  Optimizing the concentration takes a few runs and the concentration can be different for each instrument in the lab.

Illumina MiSeq “Cluster Generation by Bridge Amplification”

Illumina’s method of template preparation is termed cluster generation by bridge amplification and actually takes place on the MiSeq a step before the sequencing step.  The multiple copies are created in close proximity to each other, just as with clonal amplification, but instead of using a separate ISP for each specimen, a separate location on the flow cell is used.  A flow cell is essentially a glass slide that has universal primer anchored all over it.   This universal primers are, again, complimentary to the adapters added during the library preparation.  The combined libraries are flowed over the slide at the beginning of the run and they anneal to the universal primer.  The fragment then folds over and anneals to the second universal primer.  This strand is then replicated.  After replication, the strands are denatured creating two single strands.  These then replicate again, thus producing a cluster of the same fragment in a localized area on the slide.  This occurs for each specimen’s amplicons all over the slide.  At the end of the cluster generation step, the reverses are all cleaved off leaving only the single stranded forwards ready for sequencing.

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(https://www.illumina.com/science/technology/next-generation-sequencing/sequencing-technology.html)
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Top View of Flow Cell After Cluster Generation – Each color represents one amplicon of one specimen

Concentration is just as important in this setup as in the Ion Torrent setup.  If the concentration is too high with this assay, the clusters generated will be too close together on the flow cell, thus the sequencing signal from each cluster will overlap.  The data generated from these areas will not be able to be discerned so it will get thrown out.

Join me next quarter for the next installment – sequencing!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

Library Preparation – The First Step in a NGS Setup

Welcome back! Last quarter we discussed why Next Generation, or Massively Parallel, Sequencing is the next big thing in the world of Molecular Diagnostics. The sensitivity, the depth of coverage and the ability to interrogate many different areas of the genome at the same time were just a few of the benefits of these types of assays. Next, I would like to describe a couple different methods of library preparation, which is the first step necessary to run an NGS assay.

First of all, let’s define “Library.” I find this is the most common question technologists new to this technology ask. Essentially, a library is a specimen’s collection of amplicons produced by the assay that have been barcoded, tagged with appropriate platform adapters and purified. These will serve as the input for the next part of the NGS workflow, clonal amplification (the topic of next quarter’s blog!).  How these libraries are prepared differ depending on platform (i.e, Ion Torrent vs. MiSeq), starting material (RNA vs. DNA), and type of assay (targeted amplicon vs. exome).

Before we begin the library prep discussion, a note about the input specimen. The DNA must be quantitated using a method that is more specific than spectrophotometry – it must be specific for double-stranded DNA. It will lead to an overestimation of the amount of DNA in the specimen, which will lead to over-dilution and consequently, lower quantity of final library. Real-time PCR and a double-stranded kit with fluorometry are two examples of assays that will give accurate concentrations of double-stranded DNA.

Our lab has begun using NGS for some of our oncology assays, so I will focus on the two types we perform currently, but keep in mind, there are many other types of assays and platforms.

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Image 1: ion torrent amplicon library preparation. Source: Ion AmpliSeqTM Library Preparation User Guide – MAN0006735, Rev. 10 September 2012.

The assay we use for our Ion Torrent platform is a PCR amplicon based assay. The first step is to amplify up the 207 regions over 50 genes that contain hotspots areas for a number of different cancer types. This all occurs in one well for each specimen. Once those areas are amplified, the next step is to partially digest the primer sequences in order to prepare the ends of amplicons for the adapters necessary for the sequencing step. As shown in the figure above, two different combinations of adapters may be used. The top one, listed as the A adapter (red) and the P1 adapter (green), would be used if only one specimen was to be sequenced on the run. The A and P1 adapters provide universal priming sites so that every amplicon of every sample can be primed with the same primers, rather than having to use gene specific primers each time. The second possibility is listed below that, with the same P1 adapter (green) and a Barcode Adapter labeled X (red and blue) – it still contains the A adapter necessary for sequencing (red), but it also contains a short oligonucleotide sequence called a “barcode” (blue) that will be recognized during the analysis step based on the sequence. For example, Barcode 101’s sequence is CTAAGGTAAC – this will be assigned to specimen 1 in the run and all of the amplicons for that specimen will be tagged with this sequence. Specimen 2 will have the barcode 102 (TAAGGAGAAC) tag on all of its amplicons. During analysis, the barcodes will be identified and all of the reads with the 101 sequence will be binned together and all of the reads with the 102 sequence will be binned together. This allows many specimens to be run at the same time, thus increasing the efficiency of NGS even more. Lastly, the tagged amplicons are purified and normalized to the same concentration.

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Image 2: MiSeq amplicon library preparation. Image source: https://www.illumina.com/content/dam/illumina-marketing/documents/applications/ngs-library-prep/for-all-you-seq-dna.pdf

The assay we use for our MiSeq platform is a hybridization followed by PCR amplicon based assay. The first step is to hybridize probes to 568 regions over 54 genes that contain hotspots for a number of different cancer types. This occurs in one well for each specimen. Once the probes have hybridized, the unbound probes are washed away using a size selection filter plate. Next, the area between the probes is extended and ligated so that each of the 568 amplicons are created. These are then amplified in a PCR step using primers that are complimentary to a universal priming site on the probes, but also contain adapters plus the two indices required for paired end sequencing (the Ion Torrent platform utilizes single-end sequencing – this will be discussed in the sequencing portion in an upcoming blog!). As in the previous method, after PCR, these tagged amplicons are purified and normalized to the same concentration in preparation for the next step – clonal amplification.

Stay tuned for next quarter’s post – clonal amplification!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

Massively Parallel – the Next Generation of Sequencing

Sounds like a good title for a sci-fi novel, right?  What is the big deal about Next Generation Sequencing (NGS)?  Otherwise known as massively parallel sequencing or high throughput sequencing, NGS has become a technique used by many molecular labs to interrogate multiple areas of the genome in a short amount of time with high confidence in the results.  Throughout the next few blogs, we’ll discuss why NGS has become the next big thing in the world of molecular.  We’ll go through the steps of setting up the specimens to prepare them to be sequenced (library preparation), what types of platforms are available and what technologies they use to sequence.  Lastly, we’ll go through some of the challenges with this type of technology.

Let’s start with a review of dideoxy sequencing, otherwise known as Sanger sequencing, which has been the gold standard since its inception in 1977.  A typical setup in our lab for this assay begins with a standard PCR to amplify a region of the genome that we are interested in, say PIK3CA exon 21, specifically amino acid 1047, a histidine (CAT).  The setup would include primers complementary to an area around exon 21, a 10x buffer, MgCl2, a deoxynucleotide mix (dNTP’s), and Taq polymerase.  After amplification, the resulting products would be purified with exonuclease and shrimp alkaline phosphatase (SAP).  Next, another PCR would be set up using the purified products as the sample and using a similar mix as in the original amp, but with the addition of a low concentration of fluorescently labeled dideoxynucleotides.  These bases have no -OH group, so when they are incorporated into the product, amplification ceases on that strand.  Because they are present in a lower concentration, the incorporation of these is random and will occur at each base in the strand eventually.  The resulting products are then run and analyzed on a capillary electrophoresis instrument that will detect the fluorescent label on the dideoxynucleotides at the end of each fragment.  Shown below is an example of the output of the data:

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The bases will be shown as peaks as they are read across the laser.  The base in question in the middle of the picture is, in a “normal” sequence, an adenine (A), as seen in green.  In this case, there is also a thymine (T) detected at that same location, as seen in red.  This indicates that some of the DNA in this tumor sample has mutated from an A to a T at this location.  This causes a change from a histidine amino acid to a leucine (p.His1047Leu) and is a common mutation in colorectal cancers.

So all of this looks great, right?  Why do we need to have another method since we have been using this one for so long and it works so well?  There are a few reasons:

  1. The sensitivity of dideoxy sequencing is only about 20%.  This means lower level mutations could be missed.  The sensitivity of NGS can get down to 5% or even lower in some instances.
  2. The above picture shows the sequencing in the forward direction as well as the reverse direction.  This area then has 2x coverage – we can see the mutation in both reads.  If we could get a higher coverage of this area and be able to sequence it multiple times and see that data, we could feel more confident that this mutation is real.  In our lab, we require each area has 500x coverage so that we feel sure that we have not missed anything.  The picture below displays the same sequenced area as in the dideoxy sequencing above.  This a typical readout from an NGS assay and, as you can see, this base has a total of 4192 reads, so it has been sequenced over four thousand times.  In 1195 of those reads, a T was detected, not an A.  We can feel very confident in these results due to how many times the area was covered.
  3. The steps above detailed only amplifying this one area, but with colorectal cancer specimens, we want to know the status of the KRAS, BRAF, NRAS, and HRAS genes as well as other exons in PIK3CA  Using the dideoxy sequencing method is a lot of time and effort.  NGS can cover these areas in these five genes as well as multiple other areas (our assay looks at 207 areas total) all in the same workflow

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Join me for the next installment to discover the first steps in NGS workflow!

 

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-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.