Quantify the input DNA and assess the DNA quality before beginning library preparation.
- For a 350 bp insert size, use 25 ng input gDNA. Do not use more than 50 ng gDNA.
- For a 550 bp insert size, use 75 ng input gDNA. Do not use more than 150 ng gDNA.
- Input amounts lower than those specified result in low yield and increased duplicates.
Input DNA Quantification
Use the following recommendations to quantify input DNA:
- Successful library preparation depends on accurate quantification of input DNA. To verify results, use multiple methods.
- Use fluorometric-based methods for quantification, such as Qubit or PicoGreen. DNA quantification methods that rely on intercalating fluorescent dyes measure only double-stranded DNA and are less subject to the presence of excess nucleic acids.
- Do not use spectrophotometric-based methods, such as NanoDrop, that measure the presence of nucleotides and can result in an inaccurate measurement of gDNA.
- Quantification methods depend on accurate pipetting methods. Do not use pipettes at the extremes of volume specifications. Make sure that pipettes are calibrated.
Assessing DNA Quality
Absorbance measurements at 260 nm are commonly used to assess DNA quality:
- The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity; values of 1.8–2.0 are indicate relatively pure DNA.
- The presence of RNA or small nucleic acid fragments, such as nucleotides, can compromise both absorbance measurements.
- Make sure that samples are free of contaminants.
Illumina recommends using Coriell Institute DNA (NA 12878) as a positive control sample for this protocol.