Blue Chips – Troubleshooting Ion Torrent Data

Ah, the blue chip – not much fun to see after spending a day preparing the libraries and running clonal amp overnight.  There are a couple possible explanations for a blue chip, and you can figure them out by looking at the metrics of the run. 

Test Fragments

The test fragments serve as a control for the sequencing run.  They are spiked into the mixture of library ISPs before they are loaded on the chip.  These will allow you to figure out where the problem occurred if you encounter a blue chip.  If the Test Fragments are detected and are of sufficient quality, then this means the sequencing run worked and the problem most likely occurred before sequencing, during library prep or clonal amplification.  If the Test Fragments are not detected, then it could mean one of two things – one – the clonal amplification did not work for either the library or the Test Fragment ISPs, or – two – the sequencing run was somehow at fault.  Let’s take a look at both examples.

Troubleshooting a Blue Chip

In the event you see a blue chip, first, check to see what kinds of ISPs showed up after the analysis.  For the chip pictured above, there were ISPs that had product on them, as you can see in the Live category (6,475,553 ISPs or 95.3% of the ISPs, shown in the screenshot below).  This means clonal amp was successful for a small number of the library ISPs.  Next, there were also Test Fragments detected, at 433,392 ISPs or 6.7% of the total ISPs.  Scroll down to the bottom of the page, and you will see how the Test Fragments sequenced.  We like to see the Percent 50AQ17 and Percent 100AQ17 at least in the 80’s, but even still, you can see that these were detected and were sequenced.  Because of this, the sequencing run looks to be fine, so most likely the problem occurred before sequencing.  In this case, we believe the library prep did not yield the expected 100pM concentration, so the library pool was over-diluted prior to clonal amplification.  The library prep was repeated, and clonal amplification was run on the new pool of libraries, and the sequencing was successful.

In this next example, we have the other possibility.  This chip was blue as well (this is a 520 chip, instead of a 530, to explain the different sized pictures). 

First, there are only 2.7% Live ISPs, so even lower than the chip above.  But the even stranger thing was that there were 0.0% Test Fragments, and at the end of the analysis, there were absolutely no ISPs left to be analyzed, library or Test Fragment.  This was the only time we had ever seen a chip like this; generally, if we had blue chips, they were like the previous example.  We looked at our library pool quant and it was in the expected range, so we did not believe it was a library prep issue.  The sequencing initialization was successful and did not have any errors, so we did not believe it was a sequencing problem.  We repeated clonal amplification with the same library pool and had successful sequencing.  In speaking with our Field Application Scientist, it was decided it must have been a failure of one of the reagents of the clonal amplification – either a Taq was not present or something, so the clonal amplification never occurred, or something similar. 

Hopefully you will not experience too many of these blue chips, but if you do, I hope you are a little more prepared to troubleshoot!  Happy sequencing!

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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 and Personalized Genomic Patient Care

I’m sure that everyone has heard about next generation sequencing (NGS). But why exactly is it a big deal? Even though I have spent a significant amount of time at the bench performing “wet lab” basic science research and was acquainted with the term, I did not have practical hands-on experience with NGS prior to residency. It was not a readily accessible technology during my biomedical research days prior to medical school and so I did not entirely grasp the full power of this then disruptive technology until I was a pathology resident, and even more so, as an applicant for molecular genetic pathology (MGP) fellowships.

All of us should have previously learned about the “gold standard,” Sanger sequencing. This method combined irreversible dideoxynucleotide chain termination with a detection method such as gel electrophoresis, or on a larger, automated level, capillary electrophoresis. It was powerful because it allowed us to read the genomic map that directs cellular life, albeit only one sequence at a time. It served as the mainstay for more than a quarter of a century and still is employed for smaller scale sequencing or for long (>500 bp) stretches of DNA.

In the 1990’s, initial methods of massively parallel signature sequencing (MPSS) and pyrosequencing began to appear which would lay the groundwork for today’s massively parallel sequencing (MPS), also known as second generation sequencing, or more popularly as NGS. The two most commonly utilized NGS platforms to date are based on semi-conductor technology for the Ion Torrent (now Life Technologies) and reversible dye-terminator, sequencing based synthesis (SBS) technology for the Illumina platforms.

The power of NGS comes from its ability to simultaneously sequence 1 million to 43 billion short reads (400-500 bp each). The Human Genome Project took over a decade to complete and cost nearly $3 billion whereas NGS can sequence the same genome for a cost on the order of $1000’s, a cost that is further decreasing as the technology is refined. When I was working in research (eons ago), we had nitrocellulose based dot arrays where each “dot” represented multiple copies of a specific cDNA sequence and which helped us to build expression profiles for our particular area of study. This would be analogous to analog technology and NGS would now be considered a digital version.

With conventional PCR, we could only amplify one target sequence per sample reaction. The results were also only qualitative. The results only measurable after multiple cycles of denaturation, annealing, and extension as amplified product of the expected size or no amplified product. Then came along real-time or quantitative PCR (qPCR) which some people refer to also as RT-PCR (although this is a confusing term for people like me who were around before qPCR and think of RT-PCR as meaning reverse transcription PCR). The power of qPCR was that within the linear range of detection, we could now quantitate the amount of product present in real time. NGS also provides the resolution of qPCR in terms of quantitation.

So, as a technology, NGS nicely combines and refines some (but not all) characteristics of multiple technologies with large scale profiling. But for a non-molecular person, what is the relevance? Obviously, it has taken us some time to get to this point, even though the Human Genome Project was completed in 2003 (it started in 1990). We needed time for biomedical and translational research to provide us with clinical significance to the mutations and genetic aberrations NGS could identify. We also needed to develop tools to distinguish true mutations with clinical significance from benign polymorphisms present in our population and to build our bioinformatics support.

But here we are, stepping into the new frontier of personalized genomic medicine. Sure, there is a lot of hype surrounding it and these promises will take time to keep. But these are exciting times for someone like me who fell in love with the molecular dance that plays out within our cells. One of the reasons that I want to complete an MGP fellowship is to get more hands-on practical knowledge of the nuances of NGS from a technical standpoint but also to collaborate with other physicians in directing patients to the correct clinical trials and targeted treatment – and therein, is where the power of NGS really lies. For someone who may never get to meet the patient, at least for me, there’s probably no greater satisfaction than knowing you had a pivotal part in helping a patient more effectively combat their disease. Personalized genomic medicine is another step in that direction.

[12/12/2014: edited to fix a few inaccuracies. We apologize for the error.]

Chung

-Betty Chung, DO, MPH, MA is a third year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.