DNA Seq Prep
Genomic DNA-Seq Notes
Our group has constructed over 300 DNA-Seq libraries for the Illumina Genome Analyzer as of November, 2009. Templates have been diverse, including genomic DNAs from plants, BAC clones from plants, sub-genomic fractions enriched by hybridization, and pooled PCR products. The reagents we use in our preparations are listed below, as are sample prep notes.
Note: The items listed below represent the minimum order from New England Biolabs, and this sufficient to process 10 full Illumina libraries (20 simple libraries, if scaled at 0.5X volume). We use the New England enzymes in our own laboratory, but this is not an endorsement of their products; we expect that similar results could be obtained with any standard molecular biology grade enzyme source.
Step 1. Genomic DNA Fragmentation Step
• Nebulizers. Use Invitrogen K7025-05, or comparable. Package of 5 nebulizers, list price $121 each (2 needed for 10 samples).
• Nebulization Buffer. T10E1 (pH 7.5) containing 25% glycerol (from Sambrook et al., 2001)
• Note: our lab now uses a Bioruptor for shearing; this eliminates the need for nebulizers.
Step 2. End-Repair Step
• Quick Blunting™ Kit, New England Biolabs #E1201S. This kit is scaled for 20 – 25 μL reactions. It can be used as a substitute for the three enzyme cocktail used in the Illumina kit. List $70.00 (Nov. 2009)
Step 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 #M0212S, 200 units (40 μL volume; enough for 13 reactions). List $56.00 (Nov. 2009)
• dATP, 1 mM. Promega #U1240, 100 mM dATP stock.
Step 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 can be scaled up for the 50 μL Solexa reactions (= 12 reactions total). List $95.00 (Nov. 2009)
• Adapter oligo mix. See Cronn et al., 2008 for details.
Step 5. Gel Purification of the Products
• We now use low–melting point agarose (Promega V3841, or equivalent) for direct band isolation. When combined with a low-EDTA TAE running buffer (E0.1), the isolated bands can be simply melted at 65C, mixed well, and then directly added to the enrichment (PCR) step. We usually add 4 ul of the melted agarose/DNA to a 50 ul PCR reaction.
Step 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 packaged for 100 - 20 μL reactions, but can be scaled up for 50 μL reactions (= 40 reactions total). List $78.00 (Nov. 2009).
• PCR Primer 1. See Cronn et al., 2008 for details.
• PCR primer 2. See Cronn et al., 2008 for details.
All Steps – Notes and how to streamlining the process for large numbers of samples.
• Nebulization. If you must use 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 (median of ~450 bp) than the Illumina recommended settings (6 min at 32 psi).
• Isolating DNA from nebulizer (step 1). The large volumes of the nebulizer are best cleaned using methods designed for large volumes. We use the MoBio UltraClean15 kit (Mo Bio, #12100-300, $123/300 preps) to isolate the sheared DNA. The nebulized sample can be processed in one 2 ml tube, reducing the opportunity for cross contamination.
• Cleaning of DNAs from enzymatic steps (2, 3, 4, 6). Agencourt AMPure beads (#000130) are fast, efficient, and bind small primers and adapter complexes poorly (leading to better library enrichment). Processing requires a relatively expensive magnet for separation (Agencourt #29164); it’s worth every penny. AMPure beads easily scale to a 96 well format.
• Oligo note 1: Paired-end (PE) adapters are compatable with single- or paired-end sample preparation methods and sequencing kits.
• Oligo note 2: Note that adapter 1 MUST be 5’ phosphorylated, and that adapter 2 must have one or two terminal phosphorthioate bonds to make the adapter nuclease resistant. Long barcodes are possible; see Craig et al. (2008; see below)
• Oligo note 3: If you synthesize your own oligonucleotides, the cost is about $100 per double-stranded adapter (= oligo pair). Consult Craig et al., 2008, Cronn et al. 2008, or Bentley et al., 2008 for more information.
• Oligo note 4: PCR primers don't have to be phosphorylated, but they should be nuclease protected.
Per sample prep and nebulization (oligos not included): ~$55 for full reactions (10 complex genomes); $27 for half-reactions (20 simple genomes)
Per sample prep without nebulization (oligos not included): ~$26 for full reactions (10 complex genomes); $13 for half-reactions (20 simple genomes)
Illumina Paired-End Sequencing Sample Prep Instructions, Rev. A, April 2008.
Bentley DR, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 2008 456:53-9.
Craig DW, et al. Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 2008 Dec;5:887-93.
Quail MA, et al. A large genome center's improvements to the Illumina sequencing system. Nat Methods 2008 Dec;5:1005-10.
Publications Citing These Methods
- Cronn R, Liston A, Parks M, Gernandt DS, Shen R, and Mockler T. . pmid:18753151.
- Parks M, Cronn R, and Liston A. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology 2009 (in press).