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Library preparation

Fragmented library Mate-Paired library (short insertions) Mate-Paired library (long insertions) Mate-Paired library  Comments

Illumina library / SOLiD librarySOLiD library / Illumina library

Fragmented library






Adaptor ligation:
                                 #Ad_1                                                               #Ad_2
                       5'  TACACTCTTTCCCTACACGAC                                        TCGTATGCCGTCTTCTGCTTG 3'
                                                GCTCTTCCGATCT NNN46-146NNNA GATCGGAAGAGC
                                                CGAGAAGGCTAG ANNN46-146NNN TCTAGCCTTCTCG
                       3'  GTTCGTCTTCTGCCGTATGCT                                        CAGCACATCCCTTTCTCACAT 5'
                                 #Ad_2                                                               #Ad_1


Preamplification:
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'        3' TCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC 5'
                            #PCR_1                                                         #PCR 2

   |<-----------------------  58bp  ------------------------>|< 46-146bp >|<------------  34bp  ------------>|

5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT NNN46-146NNN AGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG 3'
3' TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNN46-146NNN TCTAGCCTTCTCGAGCATACGGCAGAAGACGAAC 5'

                         5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'
                                  sequencing primer

             P1 adapter                                             P2 adapter

5'    CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT NNN46-146NNN AGAGAATGAGGAACCCGGGGCAGTT  3'
3'  TTGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA NNN46-146NNN TCTCTTACTCCTTGGGCCCCGTC    5'

   5' CCACTACGCCTCCGCTTTCCTCTCTATG 3'                       3' TCTTACTCCTTGGGCCCCGTC  5'
           PCR primer 1, 28nt                                   PCR primer 2, 21nt
     |<---------------  41bp  ---------------->|< 46-146bp >|<------  23bp  ------->|
     total length: 120-170bp






Mate-Paired library (short insertions)

Adaptor ligation:
                                 #Ad_1                                                         #Ad_2PE
                       5'  TACACTCTTTCCCTACACGAC                                        GGTTCAGCAGGAATGCCGAG 3'
                                                GCTCTTCCGATCT NNN75-555NNNA GATCGGAAGAGC
                                                CGAGAAGGCTAG ANNN75-555NNN TCTAGCCTTCTCG
                     3'  GAGCCGTAAGGACGACGACTTGG                                        CAGCACATCCCTTTCTCACAT 5'
                                 #Ad_2PE                                                       #Ad_1

Preamplification:
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'        3' TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGAGCATACGGCAGAAGACGAAC 5'
                            #PCR_1                                                                         #PCR_2PE

   |<-----------------------  58bp  ------------------------>|< 75-555bp >|<--------------------------  61bp  ------------------------->|

5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT NNN75-555NNN AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 3'
3' TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNN75-555NNN TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGAGCATACGGCAGAAGACGAAC 5'

                         5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'        3' TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGC 5'
                                   sequencing primer 1                               sequencing primer 2






Mate-Paired library (long insertions)

Adaptor ligation:
                                                                        EcoP15I CAP    Int. adapter    EcoP15I CAP
                                                                          adapter                        adapter
                                                                                        Biotin
                                                                                          |
                                 Ad_1                                               CGTACATCCGCCTTGGCC                                            Ad_2PE
                        5'  ACACTCTTTCCCTACACGAC                                    GCATGTAGGCGGAACCGG                                    GGTTCAGCAGGAATGCCGAG 3'
                                                GCTCTTCCGATCT NNN27-29NNN CTGCTGTAC                   GT ACAGCAG NNN27-29NNNA GATCGGAAGAGC
                                                CGAGAAGGCTAG ANNN27-29NNN GACGACA TG                   CATGTCGTC NNN27-29NNN TCTAGCCTTCTCG
                     3'  GAGCCGTAAGGACGACGACTTGG                                    GGCCAAGGCGGATGTACG                                    CAGCACATCCCTTTCTCACA 5'
                                 Ad_2PE                                             CCGGTTCCGCCTACATGC                                            Ad_1
                                                                                               |   
                                                                                             Biotin


Preamplification:
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'                                                          3' TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGAGCATACGGCAGAAGACGAAC 5'
                            #PCR 1                                                                                                          #PCR 2PE

   |<-----------------------  58bp  ------------------------>|< 27-29bp >|< 7bp >|<------  20bp  ------>|< 7bp >|< 27-29bp >|<--------------------------  61bp  ------------------------->|

5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT NNN27-29NNN CTGCTGTAC ##################GT ACAGCAG NNN27-29NNN AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 3'
3' TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNN27-29NNN GACGACA TG################## CATGTCGTC NNN27-29NNN TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGAGCATACGGCAGAAGACGAAC 5'

                         5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'                                                          3' TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGC 5'
                                   sequencing primer 1                                                                              sequencing primer 2

Mate-Paired library

                                                          EcoP15I CAP                   EcoP15I CAP
             P1 adapter                                     adapter       Biotin          adapter                    P2 adapter
                                                                            |   
5'    CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT NNN27-29NNN CTGCTGTAC CGTACATCCGCCTTGGCCGT ACAGCAG NNN27-29NNN AGAGAATGAGGAACCCGGGGCAGTT  3'
3'  TTGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA NNN27-29NNN GACGACA TGGCATGTAGGCGGAACCGG CATGTCGTC NNN27-29NNN TCTCTTACTCCTTGGGCCCCGTC    5'
                                                                               
   5' CCACTACGCCTCCGCTTTCCTCTCTATG 3'                                  Int. adapter                           3' TCTTACTCCTTGGGCCCCGTC  5'
           PCR primer 1, 28nt                                                                                     PCR primer 2, 21nt
     |<---------------  41bp  ---------------->|< 27-29bp >|< 7bp >|<------  20bp  ------>|< 7bp >|< 27-29bp >|<------  23bp  ------->|
     total length: 152-156bp


reverse orientation of Internal adapter is also possible


5'    CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT NNN27-29NNN CTGCTGTAC GGCCAAGGCGGATGTACGGT ACAGCAG NNN27-29NNN AGAGAATGAGGAACCCGGGGCAGTT  3'
3'  TTGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA NNN27-29NNN GACGACA TGCCGGTTCCGCCTACATGC CATGTCGTC NNN27-29NNN TCTCTTACTCCTTGGGCCCCGTC    5'
                                                                            |   
                                                                          Biotin






List of oligonucleotides

name length sequence [5'-->3'] notes Ad_1 34nt TACACTCTTTCCCTACACGACGCTCTTCCGATCT   Ad_2 33nt P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG phosphorilated, 3' end blocked Ad_2PE 32nt P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG phosphorilated, 3' end blocked PCR_1 58nt AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT   PCR_2 34nt CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT   PCR_2PE 61nt CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT   Seq_1 33nt ACACTCTTTCCCTACACGACGCTCTTCCGATCT   Seq_2 37nt CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

name length sequence [5'-->3'] notes P1_1 41nt CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT P1 adapter P1_2 43nt P-ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT P1 adapter; phosphorilated, 3' end blocked P2_1 23nt CTGCCCCGGGTTCCTCATTCTCT P2 adapter P2_2 25nt P-AGAGAATGAGGAACCCGGGGCAGTT P2 adapter; phosphorilated, 3' end blocked PCR primer 1 28nt CCACTACGCCTCCGCTTTCCTCTCTATG   PCR primer 2 21nt CTGCCCCGGGTTCCTCATTCT   Eco_1 9nt P-CTGCTGTAC EcoP15I CAP adaptor; phosphorilated Eco_2 7nt P-ACAGCAG EcoP15I CAP adaptor; phosphorilated Int_1 20nt P-CGTACATBioCCgCCTTGGCCGT Int. adaptor; phosphorilated Int_2 20nt P-GGCCAAGGCGGATGTACGGT Int. adaptor; phosphorilated