Genome closure and finishing

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Genome closure and finishing
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Clone walking
clone insert (from shotgun library)
primer locations
Already sequenced
Unsequenced
sequencing primer
direction of extension
tgcatgatcgtgatcat acgtactagcactagtactgtagtcgatgcac
tgatcgatcgatcgatgctacgatgcatgct...
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PCR to close gaps
Scaffold
Design primers at these locations

• Run PCR for each pair of primers
• Primers must be in non-repeat sequences
• Primers must be as close as possible to each gap
• PCR works best if primers are less than 2kb apart

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What about physical gaps?
How to order and orient all scaffolds?
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Traditional strategy combinatorial PCR

• Design primers for each end of each scaffold
• With N/2 scaffolds and N ends, run a separate
  PCR reaction for every pair of ends
• (N choose 2) reactions N(N-1)/2
• Example 24 physical gaps, 48 ends,
• PCR experiments will be

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Multiplex PCR
actgagatatac
gttgagatataa
gcgacgctgctc
ccagcgctgttc
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Multiplexing more primers

• Up to 12 primers per tube
• If any two primers surround the same gap, they
  produce a product
• If more than 2 pairs react, we get multiple
  products
• Let N number of primers 48
• K max primers/tube 12

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POMPpipette optimized multiplex PCR

• minimize number of laboratory reactions
• with N48, number of combinatorial PCRs is 1128
• number of pipettings 2256
• POMP 28 reactions, 104 pipettings

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POMP

• Create pools of size K/2 6
• Each of N48 primers put into 1 pool
• So we create N/(K/2) 8 pools, P1...P8
• Now create multiplex PCR reactions with all pairs
  of pools
• (8 choose 2) 28 reactions needed

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POMP

• Guarantees that all primers are tested against
  all others
• Each reaction tests (12 choose 2) 66 pairs
• Protocol tests 6628 1848 pairs
• Only 1128 distinct pairs, so POMP has some
  redundancy - some pairs appear in more than one
  reaction

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Pooling primers
ACGTCGATGCATCGA
GCATGCTCGTACGAT
ATCGTGACAGTGAAC
GCATCGATGCATGT
ACGTCGATGCATCGA
GCATGCTCGTACGAT
GCATCGATGCATGT
ATCGTGACAGTGAAC
ACGTCGATGCATCGA
GCATGCTCGTACGAT
GCATCGATGCATGT
ATCGTGACAGTGAAC
ACGTCGATGCATCGA
GCATGCTCGTACGAT
GCATCGATGCATGT
ATCGTGACAGTGAAC
GCATCGATGCATGT
GCATCGATGCATGT
ACGTCGATGCATCGA
GCATGCTCGTACGAT
GCATCGATGCATGT
ATCGTGACAGTGAAC
GCATCGATGCATGT
GCATCGATGCATGT
GCATGCTCGTACGAT
ACGTCGATGCATCGA
GCATCGATGCATGT
ATCGTGACAGTGAAC
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
GCATCGATGCATGT
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Testing all Pools
P1
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How many pipette operations?

• 48 pipettings to create the pools (one for each
  primer)
• 2 pipettings to create each multiplex reaction
  mix
• Total 48 2(28) 104

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Results Streptococcus pneumoniae

• N48 primers, N/2 24 scaffolds, 24 gaps
• 19 products observed in the first experiment
• Q how many gaps closed?

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Interpreting results

• Case 1 product appears with Pi and Pj, but not
  in any other lane containing Pi
• see P2P6 or P7P8 on previous slide
• purify and sequence product directly
• Case 2 product appears in Pi and Pj and also in
  other lanes containing Pi
• thus two primers within Pi reacted
• see pool P5 on previous slide

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A Deconvoluting pool P5 eliminate each of the
six primers from the pool and run 6 standard PCRs
Answer p25 and p29
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B Pools P2 and P3 gave two products

• Could run 12 more PCRs eliminate each of 12
  primers and re-run multiplex PCR
• However, 5 of the 12 were eliminated by other
  results
• For example, primer p10 was eliminated by results
  from P1-P2

• Answers
• p11 in Pool 2 pairs with p13 in Pool 3
• p16 in Pool 3 pairs with p8 in Pool 2

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POMP requirements

• (Ideally) If no restriction on K, choose K based
  on N (where N 2 x (gaps))
• Make pools of size K/2

Reactions
Pipettings
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POMP summary for S. pneumoniae

• Out of 48 gaps, 42 were closed
• Strategy employs slightly under N/2 reactions,
  thus expected number of products per tube is 1
• This was borne out in experiments
• This became a standard lab technique at TIGR

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