BWA MEM misaligns my contigs to reference by a few bases

Solution!
Solution!

igv_snapshotreallyshiftedigv_snapshot igv_snapshot2

 

Dear all,

I have indexed the C. elegans reference genome with:

bwa index output/genome/ref/seq/celegans.fa

and then aligned my de novo assembly to the reference with:

bwa mem -t 8 -x intractg output/genome/ref/seq/celegans.fa input/assembly/celegans/hgap/bristol/assembly.fa > output/alignment/pacbio/bwa/ref/bristolAssembly.sam

I then

samtools view -bS output/alignment/pacbio/bwa/ref/bristolAssembly.sam > output/alignment/pacbio/bwa/ref/bristolAssembly.bam

samtools sort output/alignment/pacbio/bwa/ref/bristolAssembly.bam -o output/alignment/pacbio/bwa/ref/bristolAssemblySorted.bam

samtools index output/alignment/pacbio/bwa/ref/bristolAssemblySorted.bam

to visualise the alignment in IGV.

DNA Purification

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DNA Purification
 

Purifying DNA starting from a frozen C. elegans stock

Day 1: defrosting the stock

Defrost the stock into an agar plate.

Freezing a new strain of C. elegans

The strains I am working with should be isogenic but to increase the probability of working with an isogenic strain, I created
a new strain originating from a single worm. Pick a single worm into a new fresh agar NGM plate. Let it move away from the point
of deposition. Pick it again into a new fresh plate. This procedure of first picking into a plate and then into another makes sure
that no other worms or eggs are co-picked with the single worm of interest. Let the single worm populate the whole plate and
chunk the plate into 2 or more plates. This strain is now called differently, starting with SX, the code given to the Miska lab.
Freeze of of the plates.

Always freeze 5 vials. White plug in one of the 5 red caps. New strain from single worm is called
SX…. . SX is the Miska lab designation in the world. One of the tubes will be thawed tomorrow to check
that the freezing worked. One tube will be put in liquid nitrogen for long term storage. Three tubes will be
put in the -80 C freezer for mid term storage. Take a 15 ml Falcon tube, M9 medium, freezing buffer 2X.
Final volume of vial: 1 ml, so need 2.5 ml of freezing buffer. Pour 3.2 ml M9 on plate. Take 2.5 ml back with
pipetboy and 5 ml serological pipette. Put in Falcon. Add 2.5 ml freezing buffer to falcon. Pipet 1 ml of
solution in each vial. Transfer the vials into a polystyrene box. Tape the polystyrene box shut. Put the polystyrene box
in the -80 C freezer. The box allows the temperature decrease to be slower. Tomorrow, put test tube in tap water for 5 min.
Pour on plate and check that worms are alive. Put other tubes in inbox (4 tubes) and email lab manager. New strain SX…. is ready.

Day 4: pick worms into new plate

Pick a few worms into a fresh agar plate.

Day 7: bleach gravid adults

Once many gravid adults are present on the plate, bleach them and grow the eggs overnight at -20 C on a rotating wheel.

Before starting

Prepare 50 ml of bleach solution in a 50 ml Falcon tube: 4mL 10M NaOH, 3mL Sodium hypochlorite (stored in the cold room), 43mL H2O. The bleach solution can be stored at room temperature for one month.

Bleach in a tube

  1. Use C. elegans plates that have many gravid hermaphrodites (the eggs only will resist to the bleach treatment). Wash the plates with M9 solution: pipet M9 solution across the plate several times to loosen worms and eggs that are stuck in the bacteria. Collect the M9+worms solution in a 15 ml Falcon tube. Top up the volume to 14 ml M9.Tip: Adult hermaphrodites have a tendency to stick to the walls of plastic pipettes. To avoid this issue when harvesting the worms, you can: (i) add a small amount of detergent (NP40 0.005% or tween) to the M9 used to harvest the worms; or (ii) pre-wet the plastic pipette in M9+detergent; or (iii) use glass pipettes.

    Tip: Pipette gently to avoid collecting bits of agar together with the worms.

  2. Wash the animals three times (or until the worm suspension is clear of bacteria) with 14 ml M9. To do so, centrifuge the animals at 2,000 rpm for 2 minutes, discard the supernatant with the vacuum pump, re-suspend the worm pellet in 14 ml M9. After the last wash, do not re-suspend the worm pellet in M9 and proceed to step 3.
  3. Add 4 mL of bleaching solution to the worm pellet and immediately proceed to the next step.Optional: depending on the number for worms/level of contamination, you can increase the volume of bleach solution from 4 ml to 6 ml. You can also use stronger bleaching solution (2.5mL NaOH 10M, 7.5mL Bleach, 40mL H2O). The standard conditions should work most of the time, however.
  4. Vortex the tube for 6 minutes. Then quickly check the mixture under a dissecting scope (through the flacon tube). The worms should be dissolved and only eggs should be visible. If so, immediately proceed to the next step.Note: If necessary, you can vortex 2 additional minutes. Do not over bleach though! If the eggs stay too long in the bleach solution, they will die.
  5. Add some sterile M9 to the mixture of dissolved worms/eggs in bleaching solution (up to 14 ml final volume). Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump and immediately proceed to the next step.
  6. Resuspend the egg pellet in 14mL sterile M9. Vortex the mixture for five seconds, holding the tube horizontally. Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump. Repeat this step three times.
  7. After the last wash, resuspend the egg pellet in the desired volume. You can either pipette the eggs onto an NGM plate (use a glass pipette) or leave the eggs in liquid suspension (200 ul of sterile M9 recommended) and place them on the rotating wheel (20°C room) overnight (you will have synchronised L1 on the next day).

Day 8: transfer L1’s onto fresh agar plate

Day 10: freeze-crack L4 worms

  1. Harvest the worms from the plate into a 15 ml Falcon tube. Add 16 ml of M9 + Tween (not necessarily sterile) to the plate using a serological pipette and a pipetboy.
  2. Pipet up and down a few times.
  3. Transfer the wormy solution into the 15 ml Falcon tube.
  4. Centrifuge the tube at 2000 rpm for 2 min.
  5. You should observe a worm pellet and a supernatant that does not contain swimming worms.
  6. Remove the supernatant using a glass Pasteur pipette inside a non-filter tip attached to a vaccum pump.
  7. Dissolve the worm pellet in M9 up to 14 ml. The M9 solution does not need to be sterile.
  8. Repeat steps 4-7 three times.
  9. Centrifuge the tube at 2000 rpm for 2 min.
  10. Remove all the supernatant as to leave 100 ul of worm pellet.
  11. Freeze-crack the worms in the Falcon tube in liquid nitrogen for 20 seconds holding the tube with tweezers.
  12. At this stage, you have the possibility to store the tube at -80 C for later use.
  13. Leave the cracked worm pellet to defrost at room temperature.
  14. Start the DNeasy kit protocol.
  15. Add 360 ul ATL buffer to the tube.
  16. Transfer the tube to a 1.5 ml eppendorf tube.
  17. Add 40 ul proteinase K to the tube.
  18. Leave the tube overnight on a shaker (550 rpm) at 56 C.

Day 11: purify DNA

Centrifugation to get rid of debris + DNeasy protocol

  1. Centrifuge at 8000 g for 1.5 min.
  2. Transfer the supernatant to a new tube
  3. add 4 ul RNAse A
  4. vortex a bit
  5. leave 2 min at RT
  6. vortex
  7. add 400 ul AL
  8. vortex
  9. add 400 ul EtOh to tube.
  10. Vortex.
  11. load on column
  12. centrifuge 8000 g for 1 min
  13. discard flow-through and tube
  14. place column onto new tube
  15. add 500 ul AW1
  16. centrifuge 1 min at 8000 g
  17. discard flow-through and tube
  18. place column into new collection tube
  19. add 500 ul AW2
  20. centrifuge 3 min at 20,000 g
  21. discard flow-through and collection tube
  22. centrifuge 1 min at 20,000 g
  23. discard flow-through and collection tube
  24. place column into 1.5 ml tube
  25. add 100 100 mM Tris pH 8, leave 1 min at RT
  26. centrifuge 1 min at 8000 g

Nanodrop

Measure the nucleic acid concentration using Nanodrop.

Lift pedestal.
Clean sample area.
Drop pedestal.
Touch screen.
-> Nucleic Acids -> dsDNA

Instrument self-test…
Lift pedestal.
Clean pedestal.
Load blank.
Lower arm.

Export Nanodrop results onto USB stick.

Qubit

Measure the dsDNA concentration using Qubit.
Use low sensitivity dye (BR: broad range) dilute 200x reagent in buffer to give working solution prepare two
standards tubes in Qubit tubes

  1. standard 1: 190 ul working solution + 10 ul standard aliquot
  2. standard 2: 190 ul working solution + 10 ul standard aliquot
  3. sample: 190 ul working solution + 10 ul sample
    Qubit results for sample: X ng/ul
    Export Qubit results onto USB stick.

Agarose gel

Agarose gel: 250 ml flask. Add 0.45 g agarose in 60 ml TAE in measuring cylinder.
Tape container. Put comb in container. Microwave 1 min. Add 0.6 ul ETBR to solution.
Stir. Pour in 4 C room. Add water in flask. Stir. Pour through white material for ETBR. Wash flask with water.
Remove tape. Put gel in container. Add 5 ul ladder. Add 2 ul loading buffer to 5 ul DNA sample and load into gel.
Turn on machine on right. 150 mV, 400 mA, 30 min. Check run every 10 minutes.
Put gel in Imager. Open Image Lab. New Protocol.
Position gel, zoom in/out. Start Protocol. Image is ready on computer.
Invert contrast, change contrast, print image. Don’t touch the computer without gloves. It is contaminated.

Thanks to Jérémie Le Pen and others from the Miska lab for the invaluable advice.

 

Genetic testing: should you do it?

In recent years, the market of personalised genetic testing has emerged thanks to cheaper sequencing techniques. Some companies now offer genetic tests at an affordable price (£125 on www.23andme.com). All that is needed is a mouth swab that can be done at home. Send the box by post and receive the results within a few weeks. Find out about the probability of you developing diabetes, hypertension and dementia and take measures to prevent these diseases. Find out now whether you are one of those unfortunate people who will develop Huntington’s disease in their late years and plan your life accordingly. Why is this technique not used in any healthcare system yet? What are a hundred pounds compared to all the benefits promised by this test?
First of all, how reliable are the predictions made by the test? Given that we are dealing with increased risks, what is their magnitude and reliability? The genetic risks have been calculated in an initial population. Will they be the same in a new population? We should not remember that we are talking about predisposition and that in no case does one genetic variant always cause a disease. Secondly, once I know that I am at risk of diabetes, will I change my behaviour and lead a healthier lifestyle? Shouldn’t everyone eat healthily, exercise, avoid smoking and drink alcohol in moderate amounts? After all, healthy habits are beneficial for everybody. A recent study has shown that communicating genetic risks of disease does not reduce risky health behaviour (Hollands et al.). What is more, if a risk of incurable disease is communicated, like in the case of Huntington’s disease, imagine the unnecessary distress and sense of powerlessness that you might suffer. Thirdly, in case you are more sensitive to a drug but your healthcare system does not deal with it, what can you do with that information? Most healthcare systems in the world do not even take into account sex and weight when administering drugs, let alone genetic sequence. They do, however, adapt the treatment of breast cancer patients or leukaemia patients to their genome because the evidence of the genetic influence is very strong. Finally, do you really want to give your genetic code away? Isn’t your genetic code something special, that belongs only to you and should not be breached lightly? If you have no issue with disclosing your blueprint to anyone, what about sharing it with a health insurance company? Wouldn’t it be risky to leave your genomic sequence out there, prone to being stolen? One must admit that the probability of your data being disclosed to a health insurance company is higher than a health insurance company stealing a bit of you and sequencing you without your consent. In Switzerland and under certain conditions, health insurers can demand the results of a genetic test to be disclosed only if a test has already been carried out. In no case can a health insurance company demand from a patient to carry out a genetic test (Federal Law on Human Genetic Analysis, 2004). In case an insurance company discovers you are at a higher risk of certain diseases, they will want to charge you more for an insurance or will add reserves in the contract. It is in your interest to not deal with your genetic sequence lightly.
In conclusion, you need to ask yourself all these questions before going ahead and getting sequenced. If you deem the advantages to be higher than the downsides, then get yourself tested. I, personally, wouldn’t.

Cristian Riccio

Reference

Area under a curve

The following code illustrates the concept of integration. The concept is to divide the x-axis in segments of equal width. We then take the value of y of the function on the right side of the segment and draw a rectangle of height y. The sum of the areas of the rectangles is greater than the area under the curve and approaches the area under the curve when the width tends to 0.

Making agar bridges for electrophysiology

Ingredients

  • agarose
  • potassium chloride (KCl; MW = 74.54 g/mol)
  • glass capillaries
  • ethanol burner
  • syringe

Procedure

  1. Prepare bent capillary tubes. Alight an ethanol burner by dipping the wick in EtOH, e.g. filling a 1.5 ml tube (eppendorf) with ethanol and dipping the wick in it. Hover the capillary (at 1/3 of its length) over the fire and keep pushing on the short end with a pen until the capillary is bent at a right angle.
  2. Prepare a 20 ml solution at 1% agarose. Weight 0.2 g agarose and dissolve it in 20 ml ddH2O in a 50 ml Falcon tube.
  3. Weight the appropriate amount of KCl in order to achieve a final concentration of 3 M. I need:
    0.020 l x 3 mol/l = 0.06 mol KCl
    0.06 mol x 74.54 g/mol = 4.4724 = 4.47 g KCl
  4. DO NOT ADD THE KCl TO THE AGAROSE SOLUTION BEFORE MICROWAVING THE AGAROSE!!! (COULD CAUSE SPARKS IF KCl IS MICROWAVED)

  5. Microwave the agarose solution.
  6. Add the KCl and dissolve it.
  7. Aspire the solution with the syringe.
  8. Fill bent capillary tubes with the solution. Tip: hold the connection between capillary and syringe in order to prevent the mixture from spilling around the capillary. Quickly dry capillaries. Trim the ends of capillaries with a diamond cutter.
  9. Capillaries should not contain any bubbles.

    Storage

    Store the capillaries, i.e. agar bridges, at room temperature in a 3 M KCl solution.