Notebook 12.1: Hi-C

Chromosome conformation capture (3C) describes the structure of the genome within a cell; it's organization and structure. Better than microscopy, can tell us how close together (potentially interacting) some regions of the genome are (such as promoters and enhancers).

Hi-C: A highthroughput version of 3C is based a library preparation to build chimeric reads followed by short-read sequencing of paired-end reads. Creates a contact map of interactions correlated to spatial distance.

Notebook 12.1: Hi-C

Restriction digestion; streptavidin bead extraction; paired-seq.

Notebook 12.1: Hi-C

When a genome is digested with a restriction enzyme the genome is broken into smaller fragments. Each fragment will begin and end with a characteristic overhang of the restriction enzyme. For the restriction enzyme HindIII, the recognition site is AAGCTT, and the cut occurs between the two A's in the 5' direction (A^AGCTT) such that it leaves one A at the end a fragment, and AGCTT at the beginning of the next fragment. Let's see what this looks like:

Notebook 12.1: Hi-C

   
  def random_sequence(length):
      "return a random sequence of DNA"
      return "".join(np.random.choice(list("ACGT"), size=length))


  def restriction_digest(sequence, recognition, cut):
      """
      restriction digest a genome sequence at the given (recognition) site and
      split the site at the given position (cut) to leave overhangs. 
      """
      # cut sequence at every occurence of recognition site
      fragments = sequence.split(recognition)
      
      # add overhang that results from sequence splitting within the recognition site
      fragments = [recognition[cut:] + i + recognition[:cut] for i in fragments]
      return fragments
        

Notebook 12.1: Hi-C

  # generate a 5Mb genome
  seq = random_sequence(5000000)

  # digest the genome at every HindIII site
  fragments = restriction_digest(seq, "AAGCTT", 1)

  # print headers
  print("Restriction recognition site: A^AGCTT")
  print("Expected: [overhang-AGCTT][sequence][overhang-A]")

  # check the beginning and end of the first 10 fragments
  for i in range(10):
      print(fragments[i][:5], fragments[i][5:10], '...', fragments[i][-1:])
      

Restriction recognition site: A^AGCTT
Expected: [overhang-AGCTT][sequence][overhang-A]
AGCTT TACAA ... A
AGCTT AATGG ... A
AGCTT CCGTT ... A
AGCTT TCCCC ... A
AGCTT AGCGA ... A
AGCTT GGATT ... A
AGCTT ATATA ... A
...

Notebook 12.1: Hi-C

# get fragment lengths binned 
flens = np.histogram([len(i) for i in fragments], bins=50)

# plot distribution of fragment lengths
toyplot.bars(
    flens,
    width=400,
    height=300,
    xlabel="fragment size", 
    ylabel="number of fragments",
);
      

Action 1: Repeat for PstI enzyme: CTGCA^G

# digest the genome at every PstI site
fragments = restriction_digest(seq, "CTGCAG", 5)

# print headers
print("Expected: [overhang-G][sequence][overhang-CTGCA]")

# check the beginning and end of the first 10 fragments
for i in range(10):
    print(fragments[i][:1], '...', fragments[i][5:10], fragments[i][-5:])
    #print(fragments[i][:5], fragments[i][5:10], '...', fragments[i][-1:])
        

Expected: [overhang-G][sequence][overhang-CTGCA]
G ... TTAGC CTGCA
G ... TTCAG CTGCA
G ... CCTTA CTGCA
G ... CTTTA CTGCA
G ... GAGCT CTGCA
G ... CCCGG CTGCA
G ... ATCAC CTGCA
G ... TATGT CTGCA
G ... AATAC CTGCA
G ... TCACG CTGCA