|
Library
|
Amount added (pmol)
|
Templated ISPs (%) (successful (+)/unsuccessful (−))
|
Polyclonal population (% of total)
|
Usable population (% of total)
|
|---|
|
Ph.D.-7
|
0.325
|
54.3 (−)
|
N/A
|
N/A
|
|
Ph.D.-7
|
0.1
|
20 (+)
|
38.8
|
37.5
|
|
Ph.D.-7
|
0.075
|
11.3 (+)
|
20.4
|
59.3
|
|
Library 1
|
0.15
|
25.1 (+)
|
25.8
|
61.7
|
|
Ph.D.-7
|
0.075
|
10.4 (+)
|
40.7
|
47.2
|
|
Library 2
|
0.075
|
8.64 (+)
|
16.5
|
73.4
|
|
Library 3
|
0.075
|
6.64 (+)
|
7.4
|
72.5
|
- Ion Torrent recommends not sequencing ISPs if more than 25 % are templated because >50 % will be a polyclonal population
- The following are lists of library 1–3 structures (5′–3′) with the number of bases indicating the variable library region and the forward and reverse primers used in sequencing
-
Library 1
- GTACTCAGGAGGTTTAGT—88-bases—CCACCACCCTCATTTTC
- F-primer: CCTCTCTATGGGCAGTCGGTGAT GTACTCAGGAGGTTTAGT
- R-primer: CCATCTCATCCCTGCGTGTCTCCGACTCAG AAGAGGATTC GAAAATGAGGGTGGTGG
-
Library 2
- GAAAGGGACTGAA—51-bases—GTCAAGTTCAACAAACCC
- F-primer: CCTCTCTATGGGCAGTCGGTGAT GAAAGGGACTGAA
- R-primer: CCATCTCATCCCTGCGTGTCTCCGACTCAG AAGAGGATTC GGGTTTGTTGAACTTGACGTCAAGTTC
-
Library 3
- GGCCAGTCTGGCCAG—45-bases—GGCGGTTCTGGTGGCAGC
- F-primer: CCTCTCTATGGGCAGTCGGTGAT GGCCAGTCTGGCCAG
- R-primer: CCATCTCATCCCTGCGTGTCTCCGACTCAG AAGAGGATTC GCTGCCACCAGAACCGCC