1
$\begingroup$

I would love help outlining a basic walkthrough of the wet-lab techniques for processing blood samples from patients, all the way to loading a BeadChip, to the part just before Next Generation Sequencing takes place. Here is my basic understanding so far, that I am not 100% certain is accurate. I am not a biologist - I just communicate with them, so I do not need any confusing assay names/specifications that are important only for wet lab biologists.

Steps:
1. Collect blood from patient. 
2. Purify DNA by isolating DNA 
            (Procedure (?): lyse/sonicate, bind using ethanol, centrifuge, 
            wash with buffer, centrifuge again, rinse with water, centrifuge 
            again)
3. Cut DNA into 100-150 bp read fragments 
            (Procedure (?): with restriction enzymes? how do you determine 
            read length with restriction enzymes?)
4. Separate double stranded reads into single stranded reads 
            (Procedure (?): denature with heat) 
5. Amplify reads via PCR, leaving you with many single stranded reads
6. Disperse sample evenly into all cells on a BeadChip. The BeadChip 
      contains bountiful DNA polymerase and fluorescently tagged single nucleotides. 
7. Submit the BeadChip to the Next Generation Sequencer for processing.
8. You end up with a BUNCH of images which you can transcribe into sequences 
      based on the color illuminated in each photo capture, in each position 
      on the BeadChip.

Here's what doesn't make sense to me: Step 6. You either have to only put one PCR read product in each cell at a time - which would take FOREVER, if you wanted to sequence someone's whole genome... Or you have more than one light going off per cell, per BeadChip, in each photo. If the latter is the case (which it would have to be for high throughput wet lab experiments), how do you determine between photo captures of a given cell in a BeadChip that the nucleotide going off in photo 2 is a part of fragment n or n+k?

Does this make sense? Basically, how are BeadChips loaded with sample reads for NGS genotyping? I know that reads are "anchored", but are they anchored in each cell of the BeadChip? This is the only way this makes sense to me.

Thanks for any insights!

$\endgroup$
  • 1
    $\begingroup$ Genotyping and Next Gen Sequencing are two entirely different applications. If you are using BeadChips, you are genoptying. $\endgroup$ – swbarnes2 Oct 14 '15 at 23:30
  • $\begingroup$ The BeadChip is literally a silica chip that holds beads in an array. Each bead contains many copies of a specific oligonucleotide that bind and capture genomic DNA fragments. $\endgroup$ – canadianer Oct 15 '15 at 1:05
  • $\begingroup$ @swbarnes2, isn't the input of NGS a beadchip? What is the difference between sequencing and genotyping? NGS is a sequencing technology that gives you the nucleotide sequence of sample DNA. Is this not genotyping? $\endgroup$ – areyoujokingme Oct 17 '15 at 22:57
  • $\begingroup$ Nope. NGS puts prepared DNA fragments on a flow cell, and gives you millions of short reads. Genotyping gives you a single letter at a single pre-determined position, or in this case, the single base at a few million chosen locations. BeadArray technology is genotyping, not sequencing. $\endgroup$ – swbarnes2 Oct 19 '15 at 23:50

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.