[blueblood]

Next generation sequencing - how does it work?

by blueblood on Thu May 18, 2017 4:18 pm

I'm trying to understand next generation sequencing (NGS) and what can be learned from the results. Let me start by saying anything I write from here on out has a good chance of being 'flat out wrong', so please consider the source and verify elsewhere.

I accept there is a way to submit your DNA sample to a process that can divide your chromo­somes into many pieces. These individual pieces can be 'de-coded'. Then computer algorithms can join the many pieces in the original order to construct a model and determine your DNA profile.

I believe the results would be about 6 billion molecules (data points) for a human genome of 46 chromosomes.

I believe the cost, while decreasing over time, is still several thousand dollars.

I believe the myeloma I presented at diagnosis has a specific personal signature. Which I believe may be at a specific location in the DNA in one or more chromosomes (perhaps this is where translocations and deletions are reported in individual gene arrays).

I believe next generation sequencing is performed to help determine minimal residual disease (status) to a sensitivity great than one in a million or even one in ten million.

How does it do that?

Surely it can't map the DNA of 10 million cells.

If they know where my 'cancer signature' resides in the 6 billion+ molecules, then maybe (my assumption) when they break apart my DNA strands, they can sort the strands and only test the strands where my cancer signature might reside. Then maybe they could decode those specific stands in 10 million cells.

Finally, what do they mean when they say "less than 1 in a million"?. I don't think it means they actually tested a million cells and didn't find any. If that was the case, couldn't they run the exact same test 10 time and conclude 'less than 1 in 10 million'? Instead, it's probably how my dog, Tomahawk, can smell food when I can't - even if I inhale 10 or 100 times. So I haven't grasped the concept of sensitivity.

If by chance you do get an entire DNA map, will the data be in a form other disciplines of science could use for other purposes?

Thanks
Craig

[Multibilly]

Re: Next generation sequencing - how does it work?

by Multibilly on Fri May 19, 2017 12:00 pm

With next generation sequencing (NGS), the DNA of many millions of cells are isolated and broken up into much smaller, workable chunks. Note that the chunks are still big enough for a computer to identify where in the DNA the chunks come from, so that an overall "map" of the DNA (i.e., the genome) can be reassembled later by the computer. During this process, the areas of interest in the DNA strand from an MRD standpoint are amplified and analyzed and compared against a known good (or previously logged clonal) reference sequence. During this process, the NGS machine flags and counts areas of interest that follow a normal genetic pattern or an abnormal, mutated pattern.

Remember, in a perfect person, the DNA found in every cell of that body is identical. But with myeloma, the DNA of given lines of one or more cells may have altered DNA sequences due to disease-related mutations. So, you can't analyze just one cell's DNA when doing an MRD analysis, at least if you expect to be able to catch a mutation in a rogue cell line.

Therefore, in order to reliably detect a myeloma-related mutation of one in a million cells, you actually do need to have a sample of several million cell's DNA. To detect a mutation of one in 10 million, you would need to sample several 10's of millions of cell's DNA. By the way, MRD via flow cytometry also analyzes the various key-myeloma-related protein markers on millions of cell's surfaces. Mind blowing numbers of cells being analyzed, eh?

NGS accomplishes this magic by using massively parallel processing techniques to analyze all of these millions of small chunks of DNA strands from millions of cells at the same time.

A least that's the way I always thought it worked based on talking to one of my kids (who is a genetic microbiologist).

Last edited by Multibilly on Sat May 20, 2017 8:45 pm, edited 1 time in total.

[blueblood]

Re: Next generation sequencing - how does it work?

by blueblood on Fri May 19, 2017 2:52 pm

Multibilly,

Thanks. Realizing all DNA isn't identical due to benign and not so benign mutations, and that the next generation sequencing (NGS) might have imperfections and miss reads, I would assume they could map my entire DNA with a great deal of certainty, and that information would be available in a useful manner to determine other traits or diseases if the purpose of NGS was to map?

But since this type of NGS is specific for MRD testing (determining if something is present in a large number of DNA chains), they may be able to sort out the broken up DNA chains into a subset that they read to determine if the myeloma markers or signature are present on certain subsets. Hence not reading every chain, or for that matter reconstructing my chromosome map.

I guess I'll just have to wait until my results come back to see what they are.

[Multibilly]

Re: Next generation sequencing - how does it work?

by Multibilly on Sat May 20, 2017 6:17 pm

Craig,

I'm pretty sure that they don't sequence your entire DNA and that you don't get to walk away with an entire DNA mapping when they do myeloma MRD analysis.

Instead they tag specific loci (known areas where a gene resides in the DNA) that are responsible for managing immunoglobulins (such as the IGH and IGK genes). It is these immunoglobulin gene DNA snippets that are then sequenced and analyzed for specific clonal patterns.