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Illumina Library Preparation

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.