User guide

61
Fragment Library Preparation Using the AB Library Builder
™
System: 5500 Series SOLiD
™
Systems User Guide
AppendixC Supplemental Background Information
Preparing fragment libraries
C
After P1-T and barcoded adaptors are ligated to the sheared DNA, the library is
amplified using Library PCR Primers 1 and 2, specific to the P1 and barcoded adaptors
(see Figure 3). These primers can be used only for library amplification and not for
alternative or modified library construction adaptor design, because they do not have
3′sequences necessary for the sequencing chemistry.
Figure3 Fragment library amplification design
For RNA applications, an alternative method to generate barcoded libraries is
described in the protocols for the SOLiD
™
RNA Barcode Module 1-16 (Part
no. 4427046), SOLiD
™
RNA Barcode Module 17-32 (Part no. 4453189), and SOLiD
™
RNA Barcode Module 33-48 (Part no. 4453191).
Library PCR
Primer 2
A G A
T
T
A
G
C
A
T
A
T
G
C
A
T
G
C
G
C
A
T
A
T
C
G
C
G
C
G
G
C
G
C
G
C
G
C
C
G
A
T
G
C
T T
T C T AC T T C T C C T T G G G C C C C G T C
Library PCR Primer 2 (ss)
21 bp
5'
5'
3'
3'
P1-T Adaptor
Sheared DNA P2 Adaptor
P1-T Adaptor
Sheared DNA
Barcode--
0XX
Internal Adaptor
P2 Adaptor
Barcode--
0XX
Internal Adaptor
A T A G T G T T T T T T A T G
AT
CC C C C C C C C C C C C
CACCGACTGCCCAAG TAGAGGAAAGGG CAGGGGGG A T CT
A T A G T G T T T T T T A T G G G A G T G G T G A TCC C C C C C C C C C C C C C
Library PCR Primer 1 (ss)
28 bp
5'
3'
3'
5'
CC
Library PCR
Primer 1
Barcoded Adaptor