Midterm(Review -...

Midterm  ReviewBio5488  SP2017

Question  1  Total  coverage:2x150  bp reads  =  300  bp300  bp x  2e5  reads  =  60e6  bp

Average  coverage:60e6  bp /  12e6  bp =    5x

Poisson  Distribution!

Accurate  Call  %  (at  least  4x)=  1  – {P(0,5)  +  P(1,5)  +  P(2,5)  +P(3,5)}

Question  1  P(l)  =  0.5  

So,  5x  in  a  row  =  0.5  x  0.5  x  0.5  x  0.5  x  0.5  =0.0315

Question  5

ACmGTTCGCTTGAG

5’

3’

CTCAAGCGAACCmGT3’

5’

TGCmCAAGCGAACTC 5’3’

ACmGTTUGUTTGAG UTUAAGUGAAUCmGT

Bisulfite  Treatment

Bisulfite  Treatment

PCR/Sequencing PCR/Sequencing

ACGTTTGTTTGAG TTTAAGTGAATCGT5’ 3’

TGCAAACAAACTC3’ 5’ AAATTCACTTAGCA5’ 3’

3’ 5’

Homology

How  many  matches?  Mismatches?

How  many  gap  openings?   Extensions?  What  are  the  penalties?

Alanine GCU,  GCA,  GCC,  GCGTryptophan UGG

What  is  the  likelihood   that  W-­‐W  occurs  by  chance  in  either?

What  is  the  percent  identity  of  the  alignments?

Expression

Mixing  up  Type  I/II  error

Power  analysis

• Increasing  sequencing  depth  would  not  increase  the  statistical  power

Epigenetics

• Q3• In general: DNAme ~ DNMT3b ChIP.• BUT, for  lowly  expressed  genes,  DNAme would  not  be  that  close  to  zero.  • ALSO, DNAme in  promoter  region  should be  higher comparing  to  highly  expressed  groups

Epigenetics

• Q5:Interaction  between  Dnmt3b / methylated  DNA  and  histone  methyltransferases /  H3K36me3  (recruitment  of  one  by  another); the  associations  are  prior-­‐differentiation-­‐specific;DNA  methylation  in  gene  body  prevents  transcription  from  intragenic  alternative  TSS  (see http://www.nature.com/nature/journal/v543/n7643/full/nature21373.html)

DNA  binding  specificity

• Strong  binding  to  one  nucleotide?• Frequency:  100%

• For  any  of  the  finger,  background  frequency  is  0.25,  while  real  frequency  is  1.  So:  • Information  content  =  1.0*log2(1.0/0.25)  =  2  bits• so  10  fingers  are  necessary.

• Why  have  specificity  but  degenerate?• Allow  different  level  of  affinity  (diverse  regulatory  activity)• Allow  easy  on/off  

Technologies  used  (and  developed)  to  study  genome  folding

Adapted  from  http://web.uvic.ca/ail/equipment.htmlUnraveling  the  3D  genome:  genomics  tools  for  multiscale  exploration.  Viviana  I.  Risca  and  William  J.  Greenleaf.  Trends  in  Genetics  (2015)

Can  be  divided  into  two  broad  categories:  

1. Imaging1. Bright-­field2. Fluorescence3. EM4. Fluorescence   in-­situhybridization  (FISH),  etc.

2. Genome-­wide1. DamID2. ChIA-­PET3. Chromosome  conformation  capture-­derived,  etc.

HiC vs.  ChIA-­‐PET

• ChIA-­‐PET:  Chromatin Interaction Analysis by Paired-End Tag Sequencing

Nature Methods 6, 863 (2009)

?

Chromatin  interaction

2D  Graph  of   the  same  region