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Preparing samples for Illumina sequencing

Illumina samples

Illumina samples

Illumina samples

Normalise the concentrations of your samples

Samples submitted  should be normalised to a specific concentration and  volume depending on the method selected. Sample requirements, minimum concentrations and volumes are given for each library preparation option listed below. 

If 16 samples or fewer are submitted to the sequencing facility, the samples should be submitted in strip tubes or Eppendorf 1.5 ml Low DNA bind tubes (#0030108051). If more than 16 samples are submitted per project, samples should  be provided in an Eppendorf twin.tec® PCR plate 96 LoBind, skirted  (# 0030129512 with Eppendorf cap strips ( # 0030124839) to seal the plate. Samples should be ordered by column (vertically, eg. for 24 samples, fill the plate from A1 to H3).

All plates and tubes must be labelled with the project ID; tubes should also be labelled with the sample ID using perminant marker or freezer labels. 

Genomic sample requirements

Fragment libraries are prepared with inserts of 200-500 bp unless otherwise specified.

  • DNA should be dissolved in low TE buffer (10mM TRIS and 0.1mM EDTA, pH8) or Elution Buffer (10mM TRIS, pH8) and sent either on dry ice or packaged with ice or cool blocks.
  • Please do not send the sample lyophilised.
  • DNA samples should be free of RNA so an RNase treatment is highly recommended (treat the samples with RNAse A at the beginning of your preparation; 20 µl of a 20 mg ml-1 solution can be added during the cell pellet resuspension.
  • The amount of DNA submitted depends on the application:
    • Fragment library preparations 0.5-2.0 μg of gDNA (ideally 20 ng μl-1 in 50 μl)
    • PCR-free library preparations 2-5 μg of gDNA (min 40 ng μl-1 in 50 μl)

 

Tagmentation-based library preparation is not advised where genomes have extreme GC bias.

  • Use a lysozyme, or another gentle means of disrupting the cells prior to DNA isolation rather than using a bead based fragmentation process.
  • Use a column based clean-up such as QIAgen MiniElute/QIAquick, Zymo or Sigma Bacterial Genomic DNA extraction columns.
  • DNA should be dissolved in water or EB and not in TE buffer
  • The amount of DNA submitted depends on the application:
    • Nextera Flex 100-500 ng of gDNA (min 3.5 ng μl-1 in 30 μl) for large genomes (human) 
    • Nextera XT (for small genomes only) 5 ng of gDNA (min 0.2 ng μl-1 in 25 μl)

Damaged DNA, or samples where single-stranded DNA viruses are expected, can be sequenced using ACCEL_NGS 1S Plus DNA library preparation (Swift Biosciences) which sequences both single-stranded as well as double stranded DNA in a single preparation. It is advantageous for metagenomics, oncology/liquid biopsy, ChhIPseq, FFPE extracted DNA and ancient DNA. 

DNA fragments from 40 nucleotides can be represented in the libraries, normally fragments of 200 or 500 nucloetides are prepared unless specified.

DNA should be dissolved in low-EDTA TE (10mM Tris pH8.0, 0.1 mM EDTA)

DNA samples should be normalised to 16 ng μl-1 in a volume of 17 μl. If samples are more dilute, the lowest DNA requirement is 0.7 pg μl-1 in 17 μl.   

For low quality DNA samples, we recommend quanification by qPCR using the Alu primer pairs  (see Input DNA Quantification Assay) to accurately assess the usable amount of DNA in the samples and their integrity.

 

NB The Accel-NGS 1S Plus DNA Library Kit, adds a low complexity polynucleotide tail with an average length of 8 bases to the 3’ end of each fragment during the addition of the first NGS adapter molecule. If these tails are not trimmed bioinformatically from the sequencing data, it is normal and expected to observe them at the beginning of Read 2 (R2). When read length is close to fragment size, the tail may also be observed toward the end of Read 1 (R1) data. For specific tail trimming recommendations, please consult our Technical Note titled “Accel-NGS 1S Plus and Methyl-Seq: Tail Trimming for Better Data”.

Please supply the following with your samples:

  • DNA should be run on a 0.8-1% agarose gel to assess quality. DNA should be high molecular weight and not degraded.
  • DNA should be quantified by fluorimetry using either a Qubit fluorimeter (Life Technologies) with QUANT-iT dsDNA assay (Broad Range or High Sensitivity) or the Pico Green assay (Life Technologies).
  • Spectrophotometry methods may provide additional information but are generally inaccurate for quantitation due to the presence of small nucleic acids and contaminating chemicals.

N.B. If you are preparing your own libraries, we will rely on your quantification for sequencing. Please ensure these are accurate. Do not assume that size selection steps are sufficient to remove adaptor dimers. These small fragments will migrate with larger material and can bleed through size selection. It is imperative that qPCR and bioanalyser checks are undertaken after library construction.

Pre-made libraries 25 μl minimum at 10 nM

Transcriptomic sample requirements

  • RNA should be prepared, or at the least final preparations cleaned up, using a commercial kit such as Qiagen RNAeasy, with on-column DNase digest.
  • RNA should be supplied pure dissolved in nuclease-free water.
  • RNA should be DNAse-treated and free from chemical contaminants, especially organic solvents.
  • RNA should be sent on dry ice, ensuring there is enough dry ice to keep the samples frozen until delivery. Alternatively, when a vacuum concentrator is available RNA samples can be sent in RNAstable tubes (Biomātrica #93221-001) in moisture barrier bags with desiccants (see the Biomatrica or Cambio websites).

The amount of RNA submitted depends on the application:

  • Eukaryotic total RNA for mRNA isolation (Ribozero (human/mouse/rat) or polyA isolation available) and TruSeq stranded library preparation 0.2–2 μg of total RNA (min 2 ng μl-1 in 100 μl)
  • Bacterial total RNA for rRNA depletion and TruSeq library preparation 0.2–2 μg of total RNA (min 15 ng μl-1 in 30 μl)
  • Small RNAseq 1-2 μg total RNA or small RNAs extracted from 1-2 μg RNA
  • Pre-made libraries 25 μl minimum at 10 nM
  • RNA quality should be checked on a fragment analyser to assess the RNA fragment sizes and provide a RNA Integrity Number (RIN), RNA Integrity Score (RIS), RNA Quality Score (RQS) or RNA Quality Number (RQN); numbers which should be > 8 for vertebrate RNA (shown on the Bioanalyser result page). If a fragment analyser is not available then the RNA may be a run on a denaturing 0.8-1% agarose gel.
  • RIN numbers (or equivalents) may not be appropriate for species where the RNA fragments into nonstandard sizes, such as insects, however, the fragment analyser does not always provide accurate concentrations.
  • RNA quantity should be calculated using fluorometry, preferably using Qubit fluorometer with QUANT-iT RNA assay (Life Technology).
  • Spectrophotometry methods can provide additional information about the purity of the RNA: pure RNA should yield A260/A280 ratio of 2.0-2.2, however there is little consensus regarding the lower limit of this ratio. Chaotropic salts, carbohydrates, peptides and phenol can contribute to increased absorbance at 230 nm, but by far the most common contaminant is guanidine thiocyanate present at high concentration in extraction reagents in many RNA purification methods. A A260/A280 ratio between 2.0-2.1 indicates RNA without protein contamination.

ChIP-Seq sample requirements

Minimum 10 ng of ChIP DNA fragmented to 400-600 bp (min 0.2 ng μl-1 in 50 μl)

Amplicon sample requirements

Amplicon libraries can be prepared by ligation of adapters to gel purified amplicons or by generating the entire library using primers that contain additional 5’ sequence during PCR amplifications. PCR products should be gel purified after multiple PCR reactions (typically 4-6 reactions) and pooled for each sample. Do not assume that size selection steps are sufficient to remove adaptor dimers. These small fragments will migrate with larger material and can bleed through size selection. It is imperative that qPCR and fragment analyser checks are undertaken after library construction.

  • 1-2 μg gel-purified PCR product dissolved in 10 mM TrisHCl (pH7.5) or EB required for ligation based libraries
  • 10-100ng DNA for PCR amplified libraries

Please supply the following with your samples:

  • Check the DNA on a fragment analyser to determine the spread of the peak or region and ensure that all primers have been removed.. If a fragment analyser is not available then the DNA may be a run on a 0.8-1% agarose gel.
  • DNA should be quantified by fluorometry using either a Qubit fluorometer (Life Technologies) with QUANT-iT dsDNA assay (Broad Range or High Sensitivity) or the Pico Green assay (Life Technologies).
  • Spectrophotometry methods may provide additional information but are generally inaccurate for quantitation due to the presence of small nucleic acids and contaminating chemicals.

N.B. If you are preparing your own libraries, we will rely on your quantification for sequencing. Please ensure these are accurate.

Combined amplicon amplification and library preparation

Download a detailed protocol for 16S amplicon library preparation by PCR here.

Do not assume that size selection steps are sufficient to remove adaptor dimers. These small fragments will migrate with larger material and can bleed through size selection. It is imperative that qPCR and fragment analyser checks are undertaken after library construction.

RAD-seq

Please contact us for more information. Typically the first attempt at a project will require more material to select suitable restriction enzymes.