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lecture_notes:04-12-2010 [2010/04/16 19:54] galt |
lecture_notes:04-12-2010 [2010/04/19 03:56] (current) jmagasin |
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- | Sample Preparation {{:lecture_notes:bme235samplePrep.pdf|Nader's Powerpoint}} (Converted to PDF) | + | ====== Sample Preparation ====== |
+ | {{:lecture_notes:bme235sampleprep.pdf|Nader's Powerpoint}} (Converted to PDF) | ||
+ | |||
+ | |||
+ | //This section of the notes by Jonathan Magasin. They are keyed by title to Nader's slides.// | ||
+ | |||
+ | === Intro === | ||
+ | |||
+ | Sample preparation and creating a good libary are key to getting good reads. (At $5-10K\\ | ||
+ | in reagents per run, it matters!) Amplification is very important. We need both enough\\ | ||
+ | fragments but also to be cautious not to create a polyclonal sample. | ||
+ | |||
+ | === DNA extraction methods === | ||
+ | |||
+ | Moving to magnetic affinity resins rather than silica matrix which is currently popular. | ||
+ | |||
+ | === Organic extraction === | ||
+ | |||
+ | Lysis took around 2hrs for banana slug. | ||
+ | |||
+ | Extraction: Phenol is not used so much anymore. It is noxious and gives a nasty headache. | ||
+ | |||
+ | === Silica matrix === | ||
+ | |||
+ | Purification: Washing with higher salt solution and ethanol [//confirm//]. | ||
+ | |||
+ | The matrix principle is the same whether it is bound or not. | ||
+ | |||
+ | Extraction: Usually do two elution rounds with warm water. | ||
+ | |||
+ | === DNA measurement === | ||
+ | |||
+ | Comments on variability: | ||
+ | |||
+ | * Silica based [matrices] sometimes lose up to 80% of material. | ||
+ | * Magnetic based, lose not more than 15-20%. | ||
+ | * [Frustration with Nanodrop due to ten-fold variation.] | ||
+ | * Pipetting can lead to 50% variation, e.g. due to material on the tip. | ||
+ | * With Agilent Bioanalyzer they see 20% variation. | ||
+ | |||
+ | === Material === | ||
+ | |||
+ | The first slug they caputured had bacteria covering its skin and so they could not use it. | ||
+ | |||
+ | === Slide 13 === | ||
+ | |||
+ | The lanes smeared in a 2hr incubation. Maybe because AT-rich genome (but compared to\\ | ||
+ | //Helicobacter pylori//?!...) If they left it overnight, there was no DNA left! | ||
+ | |||
+ | === Covaris to shear the DNA === | ||
+ | |||
+ | Focuses a beam (Adapted Focused Acoustics) to shear the DNA. The water bath keeps the\\ | ||
+ | transducer cool (12°C). Very important as this is $100K equipment. Covaris advantage: no\\ | ||
+ | sequence bias [of reagents] because nothing added to the sample (just the beam required\\ | ||
+ | for shearing). | ||
+ | |||
+ | Effectiveness of cheap sample vessels discussed. | ||
+ | |||
+ | DNA digestion depends on the beam. You can tune it for desired size range, //e.g.// 300-600\\ | ||
+ | bases for Illumina. You can get up to 1000 bases with Covaris. Note however that it cannot\\ | ||
+ | get you the the roughly 3000 base fragments needed for paired ends. | ||
+ | |||
+ | With Covaris the beam can damage the DNA bases and sugar resulting in non-functional\\ | ||
+ | template. Maybe losing 30% of your mycelles due to this. A new assay being investigated \\ | ||
+ | to address this problem. | ||
+ | |||
+ | The current banana slug SOLiD run used templates generated by Covaris. | ||
+ | |||
+ | === Nebulizer === | ||
+ | |||
+ | The same thing from your local pharmacy is $5. Even if you pay $25 from .... this is still\\ | ||
+ | cheaper and simpler than Covaris. The fragment size is optimized for 454 and Illumina and\\ | ||
+ | cannot be used for SOLiD. One issue is that DNA can bind to plastic and the Nebulizer has a\\ | ||
+ | large surface area, so you're loosing at least half your material. | ||
+ | |||
+ | === Slide 19 === | ||
+ | |||
+ | We are eluting from a gel. The volume is very large so use magnetic beads to concentrate.\\ | ||
+ | They're switching to HPLC for more consistent collection/elution. | ||
+ | |||
+ | You cannot afford to lose 50% of cells if rare (//e.g.// T-cells). Imagine if you're using\\ | ||
+ | a single cell. You can't afford to lose 30-40% of genomic material. | ||
+ | |||
+ | === Sample preparation === | ||
+ | |||
+ | End repair (polishing) because you need to make blunt ends for adapter ligation. | ||
+ | |||
+ | To avoid bias, we don't want too many PCR amplifications. Check every 2 cycles for a\\ | ||
+ | visible band and then stop PCR. | ||
+ | |||
+ | They're always seeing some small fragments regardless of what part of the gel cut (5%?)\\ | ||
+ | and this causes problems in emulsion PCR. | ||
+ | |||
+ | === Rapid Library Preparation === | ||
+ | |||
+ | Requires fewer steps and less input. Built-in die (FAM standard, attached to fragments)\\ | ||
+ | so you can analyze the amount of material using a fluorometer. The yield is much less\\ | ||
+ | than standard library preparation. | ||
+ | |||
+ | === GS Rapid Library Preparation === | ||
+ | |||
+ | One bead might have a million fragments, another less, thus the 4-base key for normalization. | ||
+ | |||
+ | The 10-base barcode (MID) allows you to uniquely assign IDs to samples, so you can mix\\ | ||
+ | samples and run them in the same channel or the same physical area. | ||
+ | |||
+ | Recall that SOLiD uses very short fragments (50 bases) so you don't want your first bases,\\ | ||
+ | which are the highest quality, to be the barcode. Same for Illumina where the barcode is\\ | ||
+ | separate from the fragment. | ||
+ | |||
+ | === Rapid Library Preparation Overview -- QC === | ||
+ | |||
+ | Area under curve is the amount of material. The two spikes are the ladders. | ||
+ | |||
+ | === GS FLX Titanium Chemistry === | ||
+ | |||
+ | TissueLyser shakes the emulsion at a specified frequency. Higher frequency for smaller beads.\\ | ||
+ | One micrometer beads require shaking much faster than for the 454, but it also depends on the\\ | ||
+ | volume, tube size, shape and kind of oil. | ||
+ | |||
+ | Sequencing requires the full amount of reagents even if you do a partial run. To justify cost,\\ | ||
+ | it's better to do full runs. | ||
+ | |||
+ | === Medium volume emulsions === | ||
+ | |||
+ | Getting the best yields: Too large mycelles and you get multiple beads or fragments in them.\\ | ||
+ | However you need the mycelles large enough so there is buffer for the PCR reaction. It is a\\ | ||
+ | balance. | ||
+ | |||
+ | Oil is very viscous and you so have to be careful not to break your mycelles when dispensing\\ | ||
+ | them onto PCR plates. Nader said this is more art than science:) | ||
+ | |||
+ | === GS FLX Titanium Chemistry: Emulsion PCR === | ||
+ | |||
+ | Slide with pipetting picture insert. | ||
+ | |||
+ | The plastic caps have much better yield than ... | ||
+ | |||
+ | === GS FLX Titanium Chemistry: Emulsion PCR === | ||
+ | |||
+ | Slide with mycelles picture insert. | ||
+ | |||
+ | After PCR, take a few pictures to see what your mycelles look like. | ||
+ | |||
+ | === GS FLX Titanium Chemistry: Breaking the emulsion === | ||
+ | |||
+ | You have to wash with butanol to separate the beads from oil. Any remaining oil on the beads\\ | ||
+ | interferes with sequencing reaction. This step is time consuming, at least 2 hours and boring.\\ | ||
+ | Nader is trying to automate this (robot!). | ||
+ | |||
+ | === GS FLX Titanium Chemistry: Enrichment === | ||
+ | |||
+ | Now you need to capture just the beads with amplified DNA. If using magnetic beads, only those\\ | ||
+ | with amplification will capture the enrichment beads and get stuck to magnet. | ||
+ | |||
+ | Can use a FACs machine to sort for mono/polyclonality. (It's fast enough.) It sorts based on\\ | ||
+ | color and so can separate out beads with a single color (monoclonal) from mixed color\\ | ||
+ | (polyclonal). The dye is attached just after the primer based on 2 bases. | ||
+ | |||
+ | DNA is sheared from the beads with repeated microfluidics. Enriching for beads with good\\ | ||
+ | signal is important especially if you have long templates. | ||
+ | |||
+ | === GS FLX Titanium Chemistry: Bead deposition === | ||
+ | |||
+ | No bubbles! When you lift the pipette it can leave a bubble that will move about during\\ | ||
+ | sequencing. To avoid this you have to continue injecting material as you remove the\\ | ||
+ | pipette. (Art!) | ||
+ | |||
+ | We have Titanium in house. Our banana slug reads were all done on Titanium. |