Differences

This shows you the differences between two versions of the page.

--- lecture_notes:04-12-2010 [2010/04/16 20:14]
galt
+++ lecture_notes:04-12-2010 [2010/04/19 03:56]
jmagasin
@@ Line 1: / Line 1: @@
 ====== 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.

Banana Slug Genomics

User Tools

Site Tools

Differences

Page Tools