Preparing Samples for Illumina GAIIx Sequencing (APS)

PROTOCOL: Preparing samples for Illumina Sequencing (click to download in .docx format)

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 200µl of TE or water – target 5µg sample

-If you have never run the sample before, sonicate at least 1µg 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 µl 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 50 µl reaction:

10x T4 DNA ligase buffer (with ATP) 5 µl
10 mM dNTP mix 2 µl
T4 DNA polymerase 2.5 µl
T4 Polynucleotide kinase 2.5 µl
Klenow enzyme 1 µl
DNA sample 30 µl
Water 7 µl

2. Incubate 30 min at 20°C

3. Purify the product using the Qiagen PCR Purification Kit and elute in 32 µl 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 50µl reaction:

  • 30 µl blunted, sonicated DNA from step 3
  • 5 µl 10x Taq buffer
  • 1.5 µl 50mM MgCl2
  • 2 µl 10mM dNTPs
  • 11.5 µl DNase-free water
  • 1 µl 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 µg/ml) of your 3’A-tailed DNA using a NanoDrop spectrophotometer

2. Calculate the quantity of DNA in pmol:

Concentration (µg/ml) * Volume in ml (e.g. 0.030 ml) * 106/660 pmol/µg * 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 µM 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 as you will amplify the DNA with a PCR step.

3. Set up a ligation reaction (20 µl)

(x) pmol DNA varies
(2x) pmol Adapter varies
10x T4 ligase buffer 2 µl
T4 DNA ligase 1 µl
DNase-free water to 20 µl

4. Incubate overnight at 16°C

5. Clean-up the reaction using the Qiagen gel purification kit and elute in 32 µl water.


Note: At this point the library is adapted for Illumina sequencing and can be PCR-amplified using OLJ139 and OLJ140 as primers, available from the Tufts core, prior to submission (described below).  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 5 µl
10 mM dNTPs 2 µl
30 µM forward primer (OLJ139) 1 µl
30 µM reverse primer (OLJ140) 1 µl
10x Taq buffer 5 µl
MgCl{)2() 10 µl
H2O to 50 µl

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


Illumina01OPTIONAL 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 (50µl in a PCR tube):

Ligated DNA from step 4 5 µl
10 mM dNTPs 2 µl
30 µM forward primer 1 µl
30 µM reverse primer 1 µl
10x Taq buffer 5 µl
MgCl2 10 µl
H2O to 50 µl

b. Amplify with the same parameters as step 5(2) in the above protocol

c. Transfer 5 µl to a new tube and set up another reaction:

Ligated DNA from step 4 5 µl
10 mM dNTPs 2 µl
30 µM OLJ139 1 µl
30 µM OLJ149 1 µl
10x Taq buffer 5 µl
MgCl2 10 µl
H2O to 50 µl

3. Run on a gel and save a picture of the gel to submit with the samples. Use a NanoDrop to quantify the PCR product


Illumina02OPTIONAL 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, ligation. 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 when you design primers.