Illumina Sample Prep

Ver. 1/2009, Cronn & Liston Labs Reagents for SE or PE sample prep using PE‐compatible adapters

NOTE: The Solexa/Illumina sample prep kit is designed to process 10 DNA templates. The items listed below represent the minimum quantity available from New England Biolabs. For all items, this is enough to process 10 samples, although the minimum order will be sufficient to process far more than 10 reactions. For this reason, the cost per sample drops as you prepare additional templates.

Individual Illumina PE Sample Prep Reagents

1. Genomic DNA Fragmentation Step • Nebulizers o Invitrogen K7025‐05. Package of 5 nebulizers, list price $121 each (2 packs needed for 10 nebulizers). • Nebulization Buffer. T10E1 (pH 7.5) containing 25% glycerol (from Sambrook et al., 2001)

2. End‐Repair Step • Quick Blunting™ Kit. New England Biolabs #E1201S. This kit is scaled for 20 – 25 μl reactions; simply substitute it for the three enzyme cocktail. List $70.00 ($56.00 USFS price)

3. Addition of an ‘A’ Base to the 3’ End of the DNA Fragments • Klenow exo‐ (3′ -> 5′ exo minus), 5 U/μl. New England Biolabs

1. M0212S, 200 units (40 μl volume; enough for 13 reactions). List

$56.00 ($44.80) • Klenow buffer (provided with Klenow exo‐). • dATP, 1 mM. From Promega #U1240, 100 mM dATP stock.

4. Ligation of Adapters to the Ends of the DNA Fragments • DNA ligase (1 U/μl) and DNA ligase buffer (2X). We used the New England Biolabs #M2200S Quick Ligation™ kit. This kit is scaled for 30 ‐ 20 μl reactions, but these modified for 50 μl Solexa reactions (= 12 reactions total). List $95.00 ($76.00) • Adapter oligo mix. See Adapter/Primer page.

5. Gel Purification of the Products • nothing additional needed

6. Enrichment of the Adapter‐modified DNA Fragments by PCR • Phusion Flash DNA polymerase 2X mix (Finnzymes Oy). New England Biolabs #F‐548S. This reagent is scaled for 100 ‐ 20 μl reactions, but can be scaled for 50 μl reactions (= 40 reactions total). List $78.00 ($62.40).

• PCR Primer 1.1. See Adapter/Primer page. • PCR primer 2.1. See Adapter /Primer page.

All Steps – Streamlining the process for large numbers of samples.

• Nebulization. If you are stuck using nebulizers, you can make a multi‐sample manifold (diagram upon request) to process several samples at once. We’ve found that 2 minutes at 45 psi N2 gives a better shear distribution (smaller, with a median of ~450 bp).

• Isolating DNA from nebulizer (step 1). The large volumes of the QIAgen PCR clean‐up columns are messy and invite cross-contamination. We use the MoBio UltraClean15 kit (Mo Bio, #12100‐300, $123/300 preps) so that the entire nebulized sample can be processed in a single 2 ml tube.

• Cleaning of DNAs from enzymatic steps (2, 3, 4, 6). The GenScript “QuickClean” 96 well PCR purification kit (part #L00238, $295; $0.77 per cleaning) saves significant time and money relative to QIAgen columns. Alternatively, Agencourt AMPure beads (#29152, $667; $0.50 per cleaning) are cost effective but require an expensive magnet for separation (Agencourt #29164; $520)

• Band Isolation. Use a low‐EDTA TAE buffer (E0.1) in combination with low‐melt agarose (Promega V3841 or equivalent). After visualization, you can cut the bands from the gel, melt them at 65C, mix them well, then simply add 2 ul of the melted agarose/DNA directly to a 25 or 50 ul PCR reactions.

Illumina PE Adapters and Primers

1. Adapters (step 4) • Original Illumina PE Adapter Oligos

PE Adapter Oligo 1: /5Phos/GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE Adapter Oligo 2: 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATC*T

• Modified PE Adapters with 16 barcodes

PE_ADAPT1_ACGT: /5Phos/cgtaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_ACGT: 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTACG*T

PE_ADAPT1_CGTT /5Phos/acgaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_CGTT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGT*T

PE_ADAPT1_GTAT /5Phos/tacaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_GTAT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTA*T

PE_ADAPT1_TACT /5Phos/gtaaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_TACT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAC*T

PE_ADAPT1_AGCT /5Phos/gctaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_AGCT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGC*T

PE_ADAPT1_CTGT /Phos/cagaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_CTGT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG*T

PE_ADAPT1_GATT /5Phos/atcaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_GATT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTGAT*T

PE_ADAPT1_TCAT /5Phos/tgaaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_TCAT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCA*T

PE_ADAPT1_GGGT /5Phos/cccaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_GGGT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGG*T

PE_ADAPT1_CCCT /5Phos/gggaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_CCCT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCC*T

PE_ADAPT1_GCTT /5Phos/agcaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_GCTT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCT*T

PE_ADAPT1_TGCT /5Phos/gcaaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_TGCT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGC*T

PE_ADAPT1_AACT /5Phos/gttaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_AACT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAC*T

PE_ADAPT1_ATGT /5Phos/cataGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_ATGT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTATG*T

PE_ADAPT1_TTGT /5Phos/caaaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_TTGT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTG*T

PE_ADAPT1_CACT /5Phos/gtgaGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

PE_ADAPT2_CACT 5'‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAC*T

All oligonucleotide sequences © 2008 Illumina, Inc. All rights reserved.

ADAPTER NOTES: Note 1: Paired End (PE) adapters can be used with SE or PE sample preparation methods and sequencing kits. They are also back‐compatible with early generation (Illumina 1G, Solexa) flow cells.

Note 2: Adapter 1 MUST be 5’ phosphorylated. Also note that all “adapt 2” have a terminal phosphorthioate bond (marked by “*”) to make the adapter nuclease resistant. Longer tags are possible; see Craig et al., Nature Methods (2008)

Note 3: This procedure can be used for anealing adapters ‐ • Dilute dry oligos in TE to make a 200 uM stock • Mix 50 ul the appropriate Adapt1 and Adapt 2 pair (= 100 ul total volume, 100 uM per oligo). • Heat to 90C for 2 minutes, then cool at 2C/min for 30 minutes. Snapcool on ice when finished. • The working concentration of the adapter mix in step 4 is 50 uM, so mix 50 ul of the anealed adapter with 50 ul of dH20. This is enough adapter for 10 templates.

Note 4: Approximate cost is $100 per adapter (= oligo pair)

2. PCR Amplification Primers (step 6) • PCR Primer PE 1.0: 5'‐AAT GAT ACG GCG ACC ACC GAGATCTAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC *T

• PCR Primer PE 2.0: 5'‐CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC *T

All oligonucleotide sequences © 2008 Illumina, Inc. All rights reserved. References: Cronn et al., Nucleic Acids Research, October 2008. Craig et al., Nature Methods, October 2008. Bentley et al., Nature, November 2008.

PRIMER NOTES:

Note1: primers do not have to be phosphorylated, but are nuclease protected.

Note 2: Procedure for diluting PCR primers ‐ • Dilute dry oligos in TE to make a 250 uM stock (10X) • Prior to setting up PCR, dilute 10X stock to 1X using water. Substitute for the standard Illumina PE PCR primers.