NGI during Covid-19 outbreak

NGI is still up and running during the Covid-19 pandemic, but we are experiencing some limitations in terms of personnel and key reagents. Each NGI node is following its respective host university recommendations and will continue operation until further notice.

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Illumina Nextera DNA Flex

Low cost library preparation option for gDNA based on bead-linked transposase. Only for full plates of samples.

Method summary

The Nextera DNA Flex library prep is based on the bead-linked transposome (BLT) complex which enables both fragmentation of genomic DNA and adding adaptor sequence to those fragments in a single step. This makes it a comparatively fast protocol which can produce libraries with a consistent yield and fragment size distribution from a wide DNA input range.

  • Genomic DNA input
  • Low cost library prep option, only available in batches of 95 samples
  • Low input (≥1 ng)
  • Enzymatic shearing
  • Not recommended for FFPE
  • Amplicons (see Special sample types)
  1. Because of how the enzymatic fragmentation works, we can only offer this method with 350 bp insert sizes.
  2. With this method of normalisation, we typically see a variation in sequence reads of 20% CV among samples in a pool.

Sample requirements

The Nextera DNA Flex protocol is compatible with DNA inputs ranging from 1—500 ng. For human DNA samples and other large complex genomes, the recommended DNA input is between 100–500 ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng (requiring more PCR cycles).

To take advantage of the built-in normalisation, the input must be above 100 ng.


MinMax
Amount100 ng500 ng
Concentration3.33 ng/µL16.7 ng/µL
Volume30 µL160 ng/µL
Sample buffer10 mM Tris, pH 8–8.5 or similar (e.g. Qiagen’s EB). MilliQ water not recommended

Make sure that the DNA sample does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the Nextera tagmentation reaction and result in unexpected library insert sizes.

Multiplexing

We currently have access to 384 (combinatorial) dual indexes which means that is the highest level of multiplexing possible.

User QC

Because fragmentation is enzymatic and there is a degree of built-in sample normalisation in this chemistry, we can skip Covaris shearing and quantification steps that add time and cost to the library preparation. By only processing full plates (including one control sample) the reagent dead volume and consumable cost is minimised. This means we can only accept sample batches of 95.

The concept of user QC is that you provide your samples within the specified range of input (100–500 ng) and libraries are prepared without us doing any sample QC. We then pool the libraries by equal volume relying on the built-in normalisation. At the same time we won’t make any guarantees regarding the variability in data yield among the samples. So the compromise is that you are responsible that your samples are provided in the proper range of concentrations; the read variability may be greater compared to when we individually quantify and dilute libraries before pooling them, but the cost will be lower.

Special sample types

FFPE

FFPE or degraded DNA as input for this this protocol is not supported by the manufacturer. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting.

Amplicons

The PCR amplicon must be larger than 150 bp, as shorter amplicons can be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of around 50 bp from each distal end is expected.

What we do with your samples

No sample QC is done other than verifying that the volume requirement is met, see the User QC section.

Library preparation

The Nextera reagent is added to the sample and the transposome-coated beads become saturated with DNA. Excess DNA is washed away and a normalising effect is achieved. Next, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter sequences to both ends of the DNA fragment, enabling dual-indexed sequencing of pooled libraries on Illumina sequencing platforms. A subsequent SPRI-based size selection then removes short and long fragments and ensures a consistent fragment size profile.

The library prep is performed using our robotic liquid handling system with a program developed in-house in close collaboration with Illumina. For 100–500 ng of input, we do 5 cycles of PCR.

For details on the protocol steps and kit chemistry see the first section and the manufacturer’s documentation [1]. NGI Stockholm has automated the library preparation with liquid handling robotics which means large sample sets can be processed rapidly with consistent results.

Library QC and sequencing

The libraries are pooled by equal volume. The concentration of the pool is measured using Qubit dsDNA HS or Quant-iT dsDNA HS and the fragment size distribution is checked using chip-based capillary electrophoresis.

Expected results

With this method of normalisation we typically see a variation in sequence reads of 20% CV among samples in a pool.

The enzymatic fragmentation should not result in any region-specific bias, such as GC content; uniform coverage across the genome is expected, at the same level as with mechanical fragmentation.

Bioinformatics

Compatible with any whole genome resequencing bioinformatics workflow.

References and links

  1. Nextera DNA Flex support documentation at Illumina
  2. Bead-Linked Transposome Technology: Ushers a Next Era in Library Prep Enabling a Normalization-Free Workflow for Whole Genome and Flexible Targeted Resequencing
Applications
Relevant Technologies
Bioinformatics Pipelines
Method Status

Service

We are routinely running this method. Please visit the Order Portal to place an order.

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