Beruflich Dokumente
Kultur Dokumente
005
-20
09
April 2009
Technical Bulletin
Genome Sequencer FLX System
Summary
This bulletin describes the use of a set of up to 151 Multiplex Identifiers (MID) with the GS FLX
Titanium chemistry. This document is intended to be an extension of Technical Bulletin 09004: Using
Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry - Basic MID Set. Libraries prepared
with these Adaptors may be multiplexed in emulsion PCR (emPCR) in order to enable sample
identification following sequencing on the Genome Sequencer FLX Instrument. This bulletin enables
much deeper multiplexing for users who wish to employ the 10-base barcode strategy provided by the
MID Adaptors.
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Note: This bulletin assumes that the user is familiar with the contents of TCB-09004: Using
Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry - Basic MID Set. Please
consult this document for specific instructions regarding the preparation of MID Adaptors as
well as for the handling of libraries made with the same.
Introduction
The information contained in this document is provided to enable users of the GS FLX Titanium
sequencing chemistry to employ Multiplex Identifier (MID)-containing adaptors for General (e.g.
Shotgun) library preparation. Please note that the GS FLX Titanium General Library Preparation Method
Manual and the GS FLX Titanium General Library Preparation Kit can be used to prepare a library without
reference to the information contained in this bulletin. If you are preparing standard, non-MID
libraries, you do not need this document.
This document describes the preparation and use of up 151 Multiplex Identier (MID) Adaptors. This
extends the Basic MID Set of ten Adaptors for users requiring greater multiplexing capabilities with the
GS FLX Titanium sequencing chemistry. These MID Adaptors may be used as a replacement for the
Adaptors provided in the GS FLX Titanium General Library Preparation Kit. These Adaptors include a 10nucleotide sequence tag on Adaptor A which is unique for each MID. When different libraries are
prepared with different MIDs, they can be amplied by emPCR and sequenced together, in a multiplex
fashion; the sequencing reads can be deconvoluted by the data analysis software after the sequencing
Run, such that the reads from each of the pooled libraries are identied by their MID tag and correctly
assigned.
ACGAGTGCGT
ACGCTCGACA
AGACGCACTC
AGCACTGTAG
ATCAGACACG
ATATCGCGAG
CGTGTCTCTA
CTCGCGTGTC
TCTCTATGCG
TGATACGTCT
CATAGTAGTG
CGAGAGATAC
ATACGACGTA
TCACGTACTA
CGTCTAGTAC
TCTACGTAGC
TGTACTACTC
ACGACTACAG
CGTAGACTAG
TACGAGTATG
TACTCTCGTG
TAGAGACGAG
TCGTCGCTCG
ACATACGCGT
ACGCGAGTAT
ACTACTATGT
ACTGTACAGT
AGACTATACT
AGCGTCGTCT
AGTACGCTAT
ATAGAGTACT
CACGCTACGT
CAGTAGACGT
CGACGTGACT
TACACACACT
TACACGTGAT
TACAGATCGT
MID-40
MID-41
MID-42
MID-43
MID-44
MID-45
MID-46
MID-47
MID-48
MID-49
MID-50
MID-51
MID-52
MID-53
MID-54
MID-55
MID-56
MID-57
MID-58
MID-59
MID-60
MID-61
MID-62
MID-63
MID-64
MID-65
MID-66
MID-67
MID-68
MID-69
MID-70
MID-71
MID-72
MID-73
MID-74
MID-75
MID-76
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TACGCTGTCT
TAGTGTAGAT
TCGATCACGT
TCGCACTAGT
TCTAGCGACT
TCTATACTAT
TGACGTATGT
TGTGAGTAGT
ACAGTATATA
ACGCGATCGA
ACTAGCAGTA
AGCTCACGTA
AGTATACATA
AGTCGAGAGA
AGTGCTACGA
CGATCGTATA
CGCAGTACGA
CGCGTATACA
CGTACAGTCA
CGTACTCAGA
CTACGCTCTA
CTATAGCGTA
TACGTCATCA
TAGTCGCATA
TATATATACA
TATGCTAGTA
TCACGCGAGA
TCGATAGTGA
TCGCTGCGTA
TCTGACGTCA
TGAGTCAGTA
TGTAGTGTGA
TGTCACACGA
TGTCGTCGCA
ACACATACGC
ACAGTCGTGC
ACATGACGAC
MID-77
MID-78
MID-79
MID-80
MID-81
MID-82
MID-83
MID-84
MID-85
MID-86
MID-87
MID-88
MID-89
MID-90
MID-91
MID-92
MID-93
MID-94
MID-95
MID-96
MID-97
MID-98
MID-99
MID-100
MID-101
MID-102
MID-103
MID-104
MID-105
MID-106
MID-107
MID-108
MID-109
MID-110
MID-111
MID-112
MID-113
MID-114
MID-115
ACGACAGCTC
ACGTCTCATC
ACTCATCTAC
ACTCGCGCAC
AGAGCGTCAC
AGCGACTAGC
AGTAGTGATC
AGTGACACAC
AGTGTATGTC
ATAGATAGAC
ATATAGTCGC
ATCTACTGAC
CACGTAGATC
CACGTGTCGC
CATACTCTAC
CGACACTATC
CGAGACGCGC
CGTATGCGAC
CGTCGATCTC
CTACGACTGC
CTAGTCACTC
CTCTACGCTC
CTGTACATAC
TAGACTGCAC
TAGCGCGCGC
TAGCTCTATC
TATAGACATC
TATGATACGC
TCACTCATAC
TCATCGAGTC
TCGAGCTCTC
TCGCAGACAC
TCTGTCTCGC
TGAGTGACGC
TGATGTGTAC
TGCTATAGAC
TGCTCGCTAC
ACGTGCAGCG
ACTCACAGAG
MID-116
MID-117
MID-118
MID-119
MID-120
MID-121
MID-122
MID-123
MID-124
MID-125
MID-126
MID-127
MID-128
MID-129
MID-130
MID-131
MID-132
MID-133
MID-134
MID-135
MID-136
MID-137
MID-138
MID-139
MID-140
MID-141
MID-142
MID-143
MID-144
MID-145
MID-146
MID-147
MID-148
MID-149
MID-150
MID-151
MID-152
MID-153
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AGACTCAGCG
AGAGAGTGTG
AGCTATCGCG
AGTCTGACTG
AGTGAGCTCG
ATAGCTCTCG
ATCACGTGCG
ATCGTAGCAG
ATCGTCTGTG
ATGTACGATG
ATGTGTCTAG
CACACGATAG
CACTCGCACG
CAGACGTCTG
CAGTACTGCG
CGACAGCGAG
CGATCTGTCG
CGCGTGCTAG
CGCTCGAGTG
CGTGATGACG
CTATGTACAG
CTCGATATAG
CTCGCACGCG
CTGCGTCACG
CTGTGCGTCG
TAGCATACTG
TATACATGTG
TATCACTCAG
TATCTGATAG
TCGTGACATG
TCTGATCGAG
TGACATCTCG
TGAGCTAGAG
TGATAGAGCG
TGCGTGTGCG
TGCTAGTCAG
TGTATCACAG
TGTGCGCGTG
Note: While the Extended MID Set has been designed to the best of our ability using all
current knowledge, not all sequences have yet been thoroughly tested in library construction. It
is possible that one or more MIDs on the list may not perform as expected. Our experience to
date with 10-base MIDs has shown that approximately one in ten sequences performs below
expectations, because of unexpected dimerization/ligation events or unanticipated PCR
amplification artifacts. Please note that MID-9 and MID-12 are only used for Standard Series,
not Titanium Series methods, and are intentionally excluded herein.
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e. It is critical to verify your design by examining the structure of the adaptor that would
result from annealing of the two oligonucleotides (including verification of proper
Watson-Crick complementary base pairing) as shown in the Appendix for MID-1.
Note that you must reverse the left-to-right sequence orientation of the Aprime
adaptor to the 3-prime to 5prime direction in this exercise:
MID-13 Adaptor A:
5- CCATCTCATCCCTGCGTGTCTCCGACTCAGCATAGTAGTG-3 Ti-MID13-A
3- AGAGGCTGAGTCGTATCATCAC-5 Ti-MID13-Aprime
4. Once ordered and received, oligonucleotides should be annealed and prepared for the
Extended MID Set Adaptors mixes according to the procedures given in the document Using
Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry - Basic MID Set.
5. Consult the document Using Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry
- Basic MID Set for instructions on library preparation, emPCR and sequencing.
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Appendix
Example: MID Oligonucleotide Sequences for Ordering
OLIGO NAME
Ti-MID1-A
Ti-MID1-Aprime
Ti-MID-B
Ti-MID-Bprime
Phosphorothioate-modified bases are not shown in this figure for ease of sequence alignment
Sequencing Key is indicated in GREEN. 10 bp MID sequence is shown in YELLOW.
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