PROTOCOL: Preparing samples for Illumina Sequencing
Adam Skibinski/Kuperwasser Lab, Created 1-18-11
Note before starting: There are many ways to adapt libraries for sequencing; the following is one that has been successful for me and is reasonably simple. The general steps will be the same but the choice of kits, reagents etc. is not particularly important.
1.
Sonication of DNA – contact Tim van Opijnen in the Camilli lab for use
1.
Take an aliquot of the sample to be sequenced and dilute in 200ul of TE or water
-If you have never run the sample before, sonicate at least 1ug for several different lengths of time (e.g. 30s, 1min, 2min) and run the product on a gel until you have fragments in the range of ~200-400bp (this is what is recommended for Illumina)
2. Sonicate the sample for ~2 min (for gDNA) or ~5 min (for plasmid DNA)
3. Purify the sonicated DNA using the Qiagen PCR purification kit and elute in
32 ul of water
2.
Blunting of sonicated fragments-Sonication leaves 3’ and 5’ overhangs in the sheared DNA which must be repaired for blunt-ended ligation with Illumina adaptors
1.
Set up the following 100 ul reaction
10x T4 DNA ligase buffer (with ATP) 5 ul
10 mM dNTP mix
T4 DNA polymerase
DNA sample
Water
2.
Incubate 30 min at 20  C
T4 Polynucleotide kinase
Klenow enzyme
2 ul
2.5 ul
2.5 ul
1 ul
30 ul
7 ul
3.
Purify the product using the Qiagen PCR Purification Kit and elute in 32 ul elution buffer
3.
3’A Tailing of Blunted Fragments- This can be done with Klenow exo
(Enzymatics) but I found that Taq polymerase worked fine for this
1.
Set up the following 50ul reaction:
30ul blunted, sonicated DNA from step 3
5 ul 10x Taq buffer
1.5
ul 50mM MgCl
2
2 ul 10mM dNTPs
11.5 ul DNase-free water
1 ul Taq polymerase (Invitrogen)

2.
Incubate at 72  C for 30 min
3.
Clean up the reaction using the Qiagen PCR Purification kit eluting in 32ul
DNase-free water
4.
Your product is now ready to be ligated to Illumina adapter oligonucleotides
4.
Ligation –This step will TA-ligate the Illumina adapter sequences (which have 3’
T overhangs) onto the 3’-A overhangs of your DNA sample
1.
Measure the concentration (in ug/ml) of your 3’A-tailed DNA using a
NanoDrop spectrophotometer
2.
Calculate the quantity of DNA in pmol:
Concentration (ug/ml) * Volume in ml (e.g. 0.030 ml) * 10 6 /660 pmol/ug * 1/N,
Where N is the number of nucleotides in base pairs (suppose an average of
300). You will need twice this quantity of adapter (in pmol) in the ligation.
The adapters are at 50 uM so calculate what volume you will need for that number of pmol. You will likely need to dilute the adapter to pipet a manageable volume. It is not necessary to ligate more than 10 pmol of the library.
3.
Set up a ligation reaction (20 ul)
(x) pmol DNA
(2x) pmol Adapter varies
10x T4 ligase buffer
T4 DNA Ligase
DNase-free water varies
2 ul
1 ul to 20ul
4.
Incubate overnight at 16  C
5.
Clean-up the reaction using the Qiagen gel purification kit and elute in 32 ul water.
Note: At this point the library is adapted for Illumina sequencing and can be PCRamplified using OLJ139 and OLJ140 as primers, available from the Tufts core, prior to submission. After PCR cut out a region of the gel between 250-450 bp and submit it along with your samples. If you want to add a base-pair “tag” to differentiate samples, however, or if you want to only analyze specific sequences within your source DNA (e.g. lentiviral inserts from the TF library) then you must design specific primers to accomplish this. See “Target amplification/tagging samples” at the end of the protocol.
5.
PCR Amplification
1.
Set up the following reaction (50ul in a PCR tube):

Ligated DNA from step 5
10 mM dNTPs
5 ul
2 ul
30 uM forward primer (OLJ139) 1 ul
30 uM reverse primer (OLJ140) 1 ul
10x Taq buffer 5 ul
MgCl
2
H
2
O
10 ul to 50 ul
2.
Use the following cycling parameters in a thermocycler
95  C for 2 min
95  C for 1 min
65  C for 1 min
72  C for 1 min
Repeat previous three steps x17
72  C 4 min
4  C hold
3. Run the final PCR-amplified product on a gel to make sure bands are in the range of 250-450 bp. Submit a picture of the gel along with the samples for sequencing to the Tufts core. Contact Kip Bodi for specifics on this

PREPARING SAMPLES FOR ILLUMINA

OPTIONAL STEP: Target amplification
Note: Target amplification enriches your library for a specific subset of sequences, for example lentiviral inserts from the TF library. This replaces the PCR amplification step (step 5) in the above protocol. I describe the process below in the context of the CMV promoter/V5 tag in pLenti inserts but a similar strategy could be used to amplify any set of DNAs with a common invariant sequence.
1.
Design primers to amplify your sequence of interest (e.g. V5 tag)
For a 5’ constant sequence (e.g. CMV promoter):
-The forward primer will consist of ~20 bp of the 3’ portion of OLJ131 and
15 bp of the 5’ constant sequence
-The reverse primer will be OLJ140 (we have it in the lab)
For a 3’ constant sequence (e.g. V5 tag):
-The forward primer will be OLJ139
-The reverse primer should be constructed by taking 15 bp of the 3’ constant
sequence followed by taking ~20bp of the 5’ portion of OLJ137, then find the reverse complement of this constructed sequence
2.
PCR amplify the production – 2 step PCR a.
Set up the following reaction (50ul in a PCR tube):
Ligated DNA from step 4 5 ul
10 mM dNTPs
30 uM forward primer
30 uM reverse primer
10x Taq buffer
MgCl
2
H
2
O
2 ul
1 ul
1 ul
5 ul
10 ul to 50 ul b.
Amplify with the same parameters as step 5(2) in the above protocol c.
Transfer 5 ul to a new tube and set up another reaction
Ligated DNA from step 4 5 ul
10 mM dNTPs
30 uM OLJ139
30 uM OLJ149
10x Taq buffer
2 ul
1 ul
1 ul
5 ul
MgCl
2
H
2
O
10 ul to 50 ul
3.
Run on a gel and save a picture of the gel to submit with the samples. Cut out a region corresponding to ~150-400 bp and gel purify. Use a NanoDrop to quantify the PCR product

TARGET ENRICHMENT

OPTIONAL STEP: “Bar-coding” samples for submission in the same lane of an
Illumina sequencing reaction
Note: 4-bp “bar-codes” can be added to your adapters to distinguish different samples submitted in the same lane of the Illumina Genome Analyzer. By identifying the bar codes in the sequence data you can determine what sample each sequence came from. This is useful if you have multiple samples of low complexity (not very many different DNAs to be sequenced) and want to save $$$ but cutting down on the number of reactions. This step replaces the ligation step (step 4) in the protocol.
We have two bar-coded oligos in the lab:
IllumAdapt_ATAT: The exact sequence of OLJ131/137 but with an additional
ATAT added to the end of OLJ131
IllumAdapt_GCGT: The exact sequence of OLJ131/137 but with an additional
GCGT added to the end of OLJ131
This will allow you to submit 3 different samples at once (untagged, ATAT, and
GCGT). If you need more than that you will need to have more bar codes synthesized. Talk to Adam about this, they are fancy oligos.
Replace these tagged adapters with the Illumina 131/137 adapter in step 4. Note that if you are doing target enrichment as well as barcoding you will need to account for the altered sequence in your PCR step.