Jeff:LabNotes/Microbiome/2012-11-13

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Preliminary Sequencing Results[edit]

  • Table below summarizes results
  • The clonal rate is much better, averaging around 50% which is good
  • The mapping rate is a little low for some of these. I will use Dr. Zhang's suggestion and using the quality trimming algorithm to see if it helps the mapping rate
  • Some of the read numbers (unfiltered) are a little low. These will turn into good libraries if resequenced at a higher depth, and I will let Alan know which libraries to resequence
  • Lane 7 failed due to an instrument error and will have to be resequenced
  • I will focus more analysis on the Alzheimer's cortex library with enough reads


' Sample Type Lane Index # Reads Mapped (%) % Clonal # Usable Reads
HL135 2041_059_Alzcbl_p Neuron, alz_cerebellum 5 34 7476700 2576033 (34.35) 79.86 518850
HL135 2041_060_alzcbl_p Neuron, alz_cerebellum 5 37
HL135 60831_061_normal_p Neuron, norm_ctx 5 35 7522321 2325921 (30.92) 51.25 1133796
HL135 60831_062_normal_p Neuron, norm_ctx 5 36 16340952 12017029 (73.54) 61.66 4607752
HL135 60831_063_normal_p Neuron, norm_ctx 6 33 12367633 8354396 (67.55) 56.66 3621045
HL135 60831_064_normal_p Neuron, norm_ctx 6 34 3870522 2470947 (63.84) 40.74 1464367
HL135 60831_065_normal_p Neuron, norm_ctx 6 35 8175228 3443631 (42.12) 33.42 2292841
HL135 60831_066_normal_p Neuron, norm_ctx 6 36 15497464 11475535 (74.05) 60.76 4502562
HL135 60831_067_normal_p Neuron, norm_ctx 7 33
HL135 60831_068_normal_p Neuron, norm_ctx 7 34
HL135 2041_069_Alzcor_p NeuN-, alz_cortex 7 39
HL135 2041_070_Alzcbl_p Neuron, alz_cerebellum 7 32
HL135 2041_071_Alzcor_p Neuron, alz_cortex 8 25 24439820 17625790 (72.12) 52.85 8311147
HL135 2041_072_Alzcor_p Neuron alz_cortex 8 26 3553386 1878510 (52.87) 48.07 975482
HL135 2041_073_Alzcor_p Neuron, alz_cortex 8 29 7112256 3957668 (55.65) 79.62 806608
HL135 2041_074_Alzcbl_p Neuron, alz_cerebellum 8 32 2499658 504487 (20.18) 44.06 282209

2041_071_Alzcor_p| Analysis

  • This library has enough reads for a proper analysis. Unfortunately, for the other 3 ALz cortex samples, each had its own issue (1 had high clonal, 1 had low mapping rate, and 1 had too few reads/can be resequenced).
  • The overall plot shows a male (correct) with a lot of variation
  • The APP and DYKR1A are on Chr21, so I will focus on that. APP is from 27,252,860 to 27,543,445 bp and DYKR1A is from 38,739,236 to 38,887,680.
  • The Chr21 plot shows a few regions of high copy number.
  • The middle region is ~100k upstream of the APP gene on the same locus: 21q21.3. Possibly this is where the promoter to the APP gene. The CYYR1 gene is in this region as well, which has an unknown function.
  • I will need to sequence more Alz cortex libraries to get a better idea, and will begin to prepare more.

File:11-13-12 allchr.jpeg File:11-13-12 21chr.jpeg


Library Construction: Microwell Amplicons [edit]

  • We want to try without EtOH precipitation and compare to normally prepared libraries
  • Cursory research has shown that LMW/HMW is very similar to NEB buffer 4. Additionally, Pol I is active in NEB buffer 4.
  • I will substitute in this buffer for the Pol I reactions and scale down the reactions
  • Use 8 amplicons with new, and 8 amplicons with old library preparation
  • I will do new and Andrew will do old prep
  • I will also use 2 positive controls with 25 ng template: 1 with new and 1 with old prep

Pol 1 treatment

  • Start with 1 uL DNA
  • Add 1.5 uL ALS, 3min
  • Put on ice and add 1.5 uL NS

New Prep

Template  4 uL    
10x buffer 4 .7 uL    
dNTP .35 uL          
N6(unmodified) .35 uL  
water .4 uL         
Pol I .5 uL         
----------------------------------
Total 7 uL         

Old Prep

Template  4 uL    
10x buffer 2 2 uL    
dNTP 1 uL          
N6(unmodified) 1 uL  
water 11.5 uL         
Pol I .5 uL         
----------------------------------
Total 20 uL         
  • 37 C for 30 min, 65C for 10 min
  • Go directly to tagmentation for new prep, EtOH for old prep

Tagmentation

DNA in water  7 uL
HMW buffer  2 uL, 
1:50 diluted enzyme  1 uL
----------------------
Total 10 uL
  • 55C for 5 min

Protease treatment

  • Add 1 uL 1:100 diluted protease
  • 10 min 50C, 20 min 70C

Klenow

  • Add 1 uL exo- klenow and 1 uL dNTP to each
  • 37C for 15 min, 75 for 20 min

1st PCR round

DNA  13 uL
KAPA Robust mix  15 uL
Orange  .6 uL
blue   .6 uL
50x SYBR .24 uL
--------------------------
Total  30 uL
  • 95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8

Second round PCR

  • Pause PCR program and add following to each
1st round PCR mix 30 uL
KAPA Robust mix 35 uL
Adapter 1 2 uL
Adapter 2 Barcode 2 uL
SYBR Green 1 .56 uL
water 30.44 uL
----------------------
Total 100 uL

1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min



Bead purfication

  • Add 100 uL beads to each, mix, and let sit at RT for 8min
  • Transfer tubes to magnetic rack and let sit for 5 min
  • Remove supernatant
  • 2x 80% EtOH wash
  • Sit for 15 min
  • Elute in 23 uL Qiagen buffer


Results

  • The positive controls both worked. However, the new protocol used 7 more cycles, implicating that there is 1000x less DNA. This means that the tagmentation is 1000x less efficient
  • I want to try 1 more test tomorrow, in which I only add .6 uL of HMW buffer to the tagmentation rxn to make the buffer conc. truly 1x. It seems that higher conc. affect the tagmentation efficiency, and the rxn is very sensitive to buffer conc.
  • Some of the microwell libraries were positive, though much fainter and most likely have high clonal rates.
  • For the libraries that Andrew prepared using the EtOH method, none of them worked. I am not sure of the cause since I did not prepare them. But something went wrong with tagmentation since mine seem to have worked. I will have to prepare future samples myself since we are in a crunch.


File:11-13-12 pcr.jpg File:11-13-12 gel1.jpg

File:11-13-12 gel2.jpg