We can sequence libraries prepared by any of the following Illumina sample preparation kits:
- TruSeq PCR-free DNA sample prep kit
- TruSeq Nano DNA sample prep kit
- TruSeq RNA sample prep kit
- TruSeq Small RNA sample prep kit
- TruSeq Custom Amplicon kit
- Nextera Sample prep kit
- NexteraXT Sample prep kit
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
- NexteraXT Library - at least 1 ng DNA at 0.2 ng/μl
- 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
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.