Sample Requirements for Library Preparation
- Nextera Library - at least 50 ng DNA at 2.5 ng/μl
- Nextera Mate Pair Library - at least 1 μg DNA at 15 ng/ul
- TruSeq PCR-free Library - at least 1 μg gDNA at 20 ng/μl for a 350 bp insert size or at least 2 μg gDNA at 40 ng/μl for a 550 bp insert size
- TruSeq Nano Library - at least 100 ng gDNA at 2 ng/μl for a 350 bp insert size or at least 200 ng gDNA at 4 ng/μl for a 550 bp insert size
- TruSeq RNA v2 Library - at least 0.1-1 μg total RNA in 50 μl
- TruSeq stranded mRNA Library - at least 0.1-4 μg total RNA in 50 μl
- Ribo-Zero rRNA removal - at least 1 μg total RNA
Input DNA Quantification
Follow these gDNA input recommendations from Illumina:
- Correct quantification of genomic DNA is essential
- The ultimate success or failure of a library preparation strongly depends on using an accurately quantified amount of input DNA.
- Illumina recommends using fluorometric based methods for quantification including Qubit or PicoGreen to provide accurate quantification for dsDNA. UV-spec based methods, such as the Nanodrop, will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA and free nucleotides, which can give an inaccurate measurement of gDNA.
- DNA quantification methods that rely on intercalating fluorescent dyes measure only double-stranded DNA and are less subject to excess nucleic acids.
Assessing DNA Quality
Absorbance measurements at 260nm are commonly used to assess DNA quality:
- The ratio of absorbance at 260nm to absobance at 280nm is used as an indication of sample purity and values of 1.8-2.0 are considered indicative of relatively pure DNA.
- Both absorbance measurements can be compromised by the presence of RNA or small nucleic acid fragments such as nucleotides.
- Genomic DNA samples should be carefully collected to ensure that they are free of contaminants.
Gel electrophoresis is a powerful means for revealing the condition (including the presence or absence) of DNA in a sample.
- Impurities, such as detergents or proteins, can be revealed by smearing of DNA bands.
- RNA, which interferes with 260nm readings, is often visible at the bottom of a gel.
- A ladder or smear below a band of interest may indicate nicking or other damage to DNA.
- Where possible, or necessary, a gel should be run to assess the condition of the DNA sample.
RNA Input Recommendations
It is important to know the quality of the RNA starting material. The fragmentation conditions in the TruSeq RNA protocols were optimized for high-quality RNA.
- Illumina does not recommend the use of low quality or degraded RNA with these protocols. Use of degraded RNA can result in low yield, over-representation of the 3' ends of the RNA molecules, or failure of the protocol.
- Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer for samples with an RNA Integrity Number (RIN) value ≥ 8.
- RNA that has DNA contamination results in an underestimation of the amount of RNA used.
- Illumina recommends including a DNase step with the RNA isolation method. However, contaminant DNA is removed during mRNA purification.