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!