Integration site mapping by high-throughput sequencing of Tol2-oligo pulldown-enriched fragments
Ashwin
A. Bhandiwad1,
Tianwei Li2,
James R. Iben2,
Steven L. Coon2,
Harold A. Burgess1
1Division
of Developmental Biology, Eunice
Kennedy Shriver National Institute of Child Health and Human Development, Bethesda,
MD, 20892
2Molecular Genomics Core, Eunice
Kennedy Shriver National Institute of Child Health and Human Development, Bethesda,
MD, 20892
Abstract
Here
we present a pulldown method and pooling strategy to map transgene
insertions generated with Tol2 retrotransposons. The pulldown method
alone works well for individual zebrafish lines, but library
preparation quickly becomes a limiting factor. To solve this, we
implemented a multiplexing strategy that allows efficient reductions
in labor and cost when mapping multiple lines. Although this
procedure is optimized for interrogating Tol2 inserts we anticipate
that a similar strategy will work for any insert given suitable
pulldown probes.
Keywords:integration mapping, enhancer trap, transgenic, tol2, zebrafish
Materials
and reagents
Procedure
Extract
high molecular weight DNA using Qiagen DNeasy Blood and Tissue Kit.
Clip
fins from an adult transgenic zebrafish and place fins into 100%
ethanol on ice. Remove ethanol and let evaporate for 5-10 min.
Extract
genomic DNA as per manufacturer’s protocol, eluting with 100 µl
Buffer AE.
Using
a wide-mouth pipet tip, repeat elution with flow-through.
Combine
equal amounts of gDNA from different fish lines into pools so that
each line appears in a unique combination of pools. For each pool of
10 fish, use 100 ng gDNA per line for a total of 1000 ng for each
pool (Table 1).
DNA
shearing and library preparation
Shear
pooled DNA using Covaris focused-ultrasonicator
We
used a Covaris S2, with the onboard DNA200 settings (microTUBE AFA
Fiber Snap-Cap, 130 µl sample volume, Intensity 5, Duty Cycle 10%,
Cycles per Burst 200, Treatment Time 180 sec)
Construct
a library from each pool using the Roche SeqCap EZ Library SR User’s
Guide (formerly the Nimblegen EZ-Cap whole exome library kit). Each
of the 10 libraries is constructed with a different index barcode
from the kit.
Enrich
for fragments containing the Tol2 sequence using the SeqCap EZ
Hybridization Kit following the manufacturer’s protocol but
replacing the exome-hybridizing oligos with our custom biotinylated
oligos (Tol2 pull-down oligo 1 and 2).
Each
instance of pulldown uses 400 attomoles of each probe.
We
combined three or four pools of 10 lines for each pulldown reaction
to minimize reagent use and sequencing costs.
Post-capture
PCR amplification and purification was performed as described in
the Roche SeqCap User’s Guide to generate libraries for
sequencing.
Sequencing
and data analysis
Combine
all pools together and sequence with Illumina MiSeq using v2
chemistry yielding about 15 million 250-bp paired-end reads (about
1.5 million reads for each of the 10 pools).
Process
sequence Bioinformatics
Trim
adapter sequence
Map
sequence to zebrafish reference genome (danRer11) using BWA
Select
regions with read depth greater than 25 with mapping quality
greater than 20.
Regions
present in more than two pools excluded as non-specific, likely
off-target sequences (Supplemental File 2)
Assign
regions present in one or two pools to zebrafish lines based the
pooling matrix. For example, a region found only in pools 2 and 6
would be from line #23 (Table 1).
Lines
that show more than one possible integration location (Figure 1)
need to be disambiguated using genomic PCR.
Table
1.
Example of combinatorial pooling strategy for genomic DNA samples.
Here
we formed 10 pools from 55 lines, with 10 lines represented in each
pool. Each line is only present in 1 or 2 pools, and no two lines are
in the same 2 pools.
See
Supplemental File 1 for pooling strategies for other numbers of
lines.
Composition
of lines in each of 10 pools
|
Pool
1
|
Pool
2
|
Pool
3
|
Pool
4
|
Pool
5
|
Pool
6
|
Pool
7
|
Pool
8
|
Pool
9
|
Pool
10
|
1
|
11
|
12
|
13
|
14
|
15
|
16
|
17
|
18
|
19
|
11
|
2
|
20
|
21
|
22
|
23
|
24
|
25
|
26
|
27
|
12
|
20
|
3
|
28
|
29
|
30
|
31
|
32
|
33
|
34
|
13
|
21
|
28
|
4
|
35
|
36
|
37
|
38
|
39
|
40
|
14
|
22
|
29
|
35
|
5
|
41
|
42
|
43
|
44
|
45
|
15
|
23
|
30
|
36
|
41
|
6
|
46
|
47
|
48
|
49
|
16
|
24
|
31
|
37
|
42
|
46
|
7
|
50
|
51
|
52
|
17
|
25
|
32
|
38
|
43
|
47
|
50
|
8
|
53
|
54
|
18
|
26
|
33
|
39
|
44
|
48
|
51
|
53
|
9
|
55
|
19
|
27
|
34
|
40
|
45
|
49
|
52
|
54
|
55
|
10
|
Figure
1:
Histogram of number of integration locations for 55 lines identified
using this pulldown protocol

Supplemental
File 1: Combinatorial
Matrices for other numbers of fish lines.
Supplemental
File 2: Common
off-target and unknown sequences obtained by pull-down protocol