MALBAC

The Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is actually a method for single-cell genomic DNA preamplification. The final library structure depends on the method of choice after the pre-amplification. MALBAC is a quasilinear preamplification method that reduces the bias associated with nonlinear amplification. During the first 5 cycles of preamplification, only the original genomic DNA and the first round of products are used as the templates, which reduces errors.

Adapter and primer sequences:

Step-by-step library generation

(1) Sort or handpick single cells, melt genomic DNA at 94 ℃, quench at 0 ℃, and the MALBAC random primers will anneal randomly to single-stranded DNA:

5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG
                               NNNNNNNN                                 NNNNNNNN                                 NNNNNNNN
3'- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX...XXXXX -5'

(2) Extension using a polylmerase with strand displacement activity such as Bst or Phi29 at 68 ℃ (will kick off the product in front):

5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG
                               NNNNNNNN-------------------------------->NNNNNNNN-------------------------------->NNNNNNNN----------->
3'- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX...XXXXX -5'

(3) There are two types of templates at this stage:

 Origincal genomic DNA:

3'- XXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXX -5'


 The Semi-amplicon:

3'- XXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXGAGGTGTGATGGAGTTGGTAGTGAGTG -5'

(4) Again, melt DNA at 94 ℃, quench at 0 ℃ for the MALBAC random primers annealing, and extension at 68 ℃ (both the original genomic DNA and the Semi-amplicon will be used as templates):

 Original genomic DNA as template (will generate new Semi-amplicon):

5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG
                               NNNNNNNN-------------------------------->NNNNNNNN-------------------------------->NNNNNNNN----------->
3'- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX...XXXXX -5'


 The Semi-amplicon as template (will generate Full-amplicon):

5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG          5'- GTGAGTGATGGTTGAGGTAGTGTGGAG
                               NNNNNNNN-------------------------------->NNNNNNNN-------------------------------->NNNNNNNN--------------...------------------------------->
3'- XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX...XXXXXGAGGTGTGATGGAGTTGGTAGTGAGTG -5'

(5) There are three types of DNA at this stage:

 Original genomic DNA:

3'- XXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXX -5'


 The Semi-amplicon:

3'- XXXXXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXXXXGAGGTGTGATGGAGTTGGTAGTGAGTG -5'


 The Full-amplicon:

5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXX...XXXCTCCACACTACCTCAACCATCACTCAC -3'

(6) Looping at 58 ℃ (only the Full-amplicon will self-anneal to a looped structure to prevent further amplification):

  XXX...XXX         
  X        CTCCACACTACCTCAACCATCACTCAC -3'
  X        GAGGTGTGATGGAGTTGGTAGTGAGTG -5'
  XXX...XXX

(6) Repeat the 0 ℃ quenching, 68 ℃ extension and 58 ℃ looping cycles for 5 times. During these cycles, the original gDNA will yield Semi-amplicons, and Semi-amplicons will yield Full-amplicons, and Full-amplicons will stay unchanged once generated.

(7) Use the MALBAC PCR primer to amplify the Full-amplicon using regular PCR conditions:

5'- GTGAGTGATGGTTGAGGTAGTGTGGAG------>
5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXXXXXXXXX...XXXXXXXXXXXXXCTCCACACTACCTCAACCATCACTCAC -3'
                                                     <------GAGGTGTGATGGAGTTGGTAGTGAGTG -5'

(8) Final product of the amplified gDNA, and this can be used as the staring material for the library construction using your favourite kits, such as TruSeq or Nextera, to make a sequencing ready library:

5'- GTGAGTGATGGTTGAGGTAGTGTGGAGXXXXXXXXXXXXX...XXXXXXXXXXXXXCTCCACACTACCTCAACCATCACTCAC -3'
3'- CTCCACACTACCTCAACCATCACTCACXXXXXXXXXXXXX...XXXXXXXXXXXXXGTGAGTGATGGTTGAGGTAGTGTGGAG -5'

(9) Final library structure (depends on the library preparation kits used, two most popular ones are shown below):

Using Nextera:

5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
    TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
           Illumina P5              i5         s5              ME                gDNA                ME               s7          i7            Illumina P7


Using TruSeq:

5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTXXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAXXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
          Illumina P5                   Truseq Read 1           gDNA            Truseq Read 2               8 bp         Illumina P7
                                                                                                           Index

Library sequencing:

Standard sequencing workflow is used, and you can check any other pages.