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lecture_notes:04-12-2010 [2010/04/16 12:58]
galt
lecture_notes:04-12-2010 [2010/04/18 20:56] (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. ​
lecture_notes/04-12-2010.1271447902.txt.gz · Last modified: 2010/04/16 12:58 by galt