Next Generation Sequencing on Illumina MiSeq
- Sample library preparation using Illumina kits
- MiSeq Sequencing
The Illumina MiSeq System combines proven sequencing-by-synthesis (SBS) technology with a workflow that enables you to go from DNA to analyzed data in as little as eight hours. The MiSeq integrates cluster amplification, sequencing and data analysis in a single instrument. For the read length and amount of output there are number of alternatives to be considered depending on needs (see section MiSeq Performance below). Also for the strategy of library construction there are many alternatives (see section MiSeq Applications below). For your convenience we urge you to register as an Illumina user (register at my.illumina.com). You will not only get access to a wealth of information, as prices for various reagents, but also to Illumina Support with whom you can discuss strategies and solutions for your project. Additionally, as an Illumina user you will also get access to the Illumina BaseSpace (https://basespace.illumina.com) service. BaseSpace is a cloud-based service and Illumina's genomics computing environment for next-generation sequencing (NGS) data analysis and management. As a sequencing run is started data is simultaneously pushed out to BaseSpace and you can almost in real-time follow the progress of your run. It also means that sequencing labs can easily and securely analyze, archive, and share sequencing data.
|Cycels – Paired-End Reads|
|50 cycle kit (2 x 25 bp) V2||5.5 hrs||750 - 850 Mb|
|150 cycle kit (2 x 75 bp) V3||20 hrs||3.3 - 3.8 Gb|
|300 cycle kit (2 x 150 bp) v2||Nano||300 Mb|
|300 cycle kit (2 x 150 bp) v2||Micro||1.2 Gb|
|300 cycle kit (2 x 150 bp) V2||24 hrs||4.5 - 5.1 Gb|
|500 cycle kit (2 x 250 bp) V2||Nano||500 Mb|
|500 cycle kit (2 x 250 bp) V2||39 hrs||7.5 - 8.5 Gb|
|600 cycle kit (2 x 300 bp) V3||55 hrs||13.2 - 15 Gb|
|Reads Passing Filter|
|V2 Chemistry||V3 Chemistry|
|Single Reads||15 - 17 M||22 - 25 M|
|Paired-End Reads||30 - 34 M||44 - 50 M|
|2 x 25 bp||> 90% bases > Q30|
|2 x 75 bp||> 85% bases > Q30|
|2 x 150 bp||> 80% bases > Q30|
|2 x 250 bp||> 75% bases > Q30|
|2 x 300 bp||> 70% bases > Q30|
*M, million; Mb, million base pairs (bp); Gb, giga bp or a billion bp
(Information courtesy of Illumina)
The MiSeq supports a wide range of applications, including the following (in all cases multiplexing ≤384 samples / sequencing run is allowed):
- de novo genome sequencing
- Targeted resequencing
- de novo transcriptome sequencing
- Small RNA sequencing
- TruSeq Custom Amplicon Sequencing
- Metagenome sequencing
Sample Requirements for Library Preparation and Library Solutions
This table presents a few different solutions for various type of library preparations. But we recommend you to check out the Illumina Library Comprehensive NGS Library Preparation Solutions, where you will find guides that will take you through different strategies based on your specific needs.
|Fragment size||Amount required||Concn. required*|
|Nextera Library||≥50 ng gDNA||≥2.5 ng/μl|
|Nextera Mate Pair Library||≥1 μg gDNA||≥15 ng/ul|
|TruSeq PCR-free Library||350 bp||≥1 μg gDNA||≥20 ng/μl|
|550 bp||≥2 μg gDNA||≥40 ng/μl|
|TruSeq Nano Library||350 bp||≥100 ng gDNA||≥2 ng/μl|
|550 bp||≥200 ng gDNA||≥4 ng/μl|
|TruSeq RNA v2 Library||0.1-1 μg total RNA in 50 μl|
|TruSeq stranded mRNA Library||0.1-4 μg total RNA in 50 μl|
* In all cases try to avoid the addition of EDTA, a 10mM Tris buffer, pH 8.5 or H2O are in most cases suitable as a solvent.
(Information courtesy of Illumina)
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, such as Qubit (www.thermofisher.com) or PicoGreen (www.thermofisher.com) to provide accurate quantification for dsDNA. UV-spectrophotometric based methods, such as ordinary spectroscopy (e.g. NanoDrop), will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA and free nucleotides, which can give an inaccurate measurement of gDNA. In contrary, 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
Genomic DNA samples should be carefully collected to ensure that they are free of contaminants. Absorbance measurements at 260nm are commonly used to assess DNA quality:
- The ratio of absorbance at 260nm to absorbance 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.
Gel electrophoresis is a powerful means for revealing the condition 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 the RNA integrity of isolated total RNA using an analysis on an Agilent 2100 BioAnalyzer and aim for samples having an RNA Integrity Number (RIN) value ≥ 8 (see this pdf).
- RNA that has DNA contamination results in an overestimation of the amount of RNA used. Therefore Illumina recommends including a DNase step with the RNA isolation method. Although, contaminant DNA and rRNA will be removed during mRNA purification
Processing more than one sample within the same sequencing run (multiplexing) requires barcoding (also called indexing) of each sample. Sample multiplexing is a useful approach when pooling various genomes, transcriptomes, metagenomes or individual amplicons within the same analysis.
To accomplish this, individual "barcode" or “index” sequences together with adaptor regions, are added to each sample so they can be distinguished and sorted during data analysis.
- Multiplex up to 384 samples per run for greater efficiency
- Get accurate bi-directional amplicon sequencing
- Get accurate and automatic sorting of data into indexed bins
For instance see the Illumina options:
- Nextera XT Index Kit (24 indices, 96 samples)
- Nextera XT Index Kit (96 indices, 384 samples)
- Nextera XT v2 Index Kit set A, B, C, or D (96 indices, 384 samples each – combine all four for 384 index combinations)
Forms for Illumina MiSeq sequencing
For each MiSeq sequencing run two different forms/files are needed from the end-user, a Sample Sheet (i) and a Sequencing Form (ii).
- The Sample Sheet (i) is a comma-separated values file (*.csv) that stores information required to set up, perform, and analyze a sequencing run. Illumina recommends that you create your sample sheet before preparing your sample libraries. You can create your sample sheet using the Illumina Experiment Manager or create it manually using a text editor, such as Excel or Notepad.
The Illumina Experiment Manager is a wizard-based application that guides you through the steps to create your sample sheet. Using the Illumina Experiment Manager not only reduces syntax errors, but also provides prompts for information that applies to your sample type and analysis workflow. It provides a feature for recording parameters for the sample plate, such as sample ID, project name, dual indices, and barcode information. Then, the sample plate information can be imported to the sample sheet using the Illumina Experiment Manager. The Illumina Experiment Manager can be run on any Windows platform. To download the software, go to the Illumina Experiment Manager Support page on the Illumina website. For more information, see the Illumina Experiment Manager User Guide (part # 15031335). The finished Sample Sheet will than need to be mailed to us, together with a Sequencing Form described in the next paragraph.
- A Sequencing Form (ii) (xlsx; 12 kB) is required, containing information about the client, invoicing information, material provided, method for library construction, and the sequencing protocol requested. Here you will also be asked to suggest a DNA concentration to be used if there is such pre-knowledge about an optimal DNA concentration for cluster generation of your sample(s). You will also be asked to suggest a fraction of PhiX to be used, if there is a pre-knowledge. The PhiX Control is a reliable, adapter-ligated library used as a control for Illumina sequencing runs. The library is derived from the small, well-characterized PhiX genome, offering several benefits for sequencing and alignment. The PhiX library provides a quality control for cluster generation, sequencing, and alignment, and a calibration control for cross-talk matrix generation, phasing, and prephasing. The PhiX DNA is spiked into your DNA sample and Illumina recommends a spike-in of PhiX at 1% for most libraries. For low-diversity libraries, such as amplicons, an increase up to at least 5% has been found beneficial. But the optimal fraction has to be found out empirically for each sample.
Prices for Library Preparation and Illumina MiSeq Sequencing
For reagents we are charged in US$ which means that prices will fluctuate over time due to currency exchange rates. As a registered Illumina user you will get the current prices on all Illumina reagents at my.illumina.com. Most kits for library construction are in at least 24x, 48x or 96x sample formats, but the most common kits can be split among users, but for some requests you will need to be prepared to pay more. As earlier mentioned, final prices will be dependent on the currency exchange rate, but estimates in SEK are given below.
Some examples of costs for library construction:
|Kit||Cost per 24 libraries||Cost per 48 libraries||Cost per 1 library|
|TruSeq DNA PCR-Free||10,900 SEK||460 SEK|
|TruSeq Nano DNA||460 SEK|
|Nextera DNA||17,700 SEK||750 SEK|
|Nextera XT (small genomes)||7,050 SEK||300 SEK|
|TruSeq RNA Kit v2||30,500 SEK||650 SEK|
|TruSeq Stranded mRNA||34,900 SEK||740 SEK|
Some examples of costs for sequencing reagents:
|Sequencing cost per run|
|500 cycle kit (2 x 250 bp) V2||9,980 SEK|
|600 cycle kit (2 x 300 bp) V3||13,500 SEK|
In addition we surcharge all users an additional 25% on costs for library construction and sequencing to cover for additional consumables, additional reagents and other needs. For all users “overhead” charges will be applied. For users outside Lund University we also need to charge for labor on an hourly basis. Please, inquirer prices!
The sequencing Facility is situated at Sölvegatan 37, in the Ecology Building on Floor 2 (stair case A)
For inquiries about Illumina MiSeq sequencing and analyses on Agilent BioAnalyzer contact:
Telephone: +46 46-222 45 49
Mobile: +46 709-57 18 04
E-mail: Tomas.Johansson [at] biol.lu.se
For inquiries about ABI Sanger sequencing contact:
Telephone: +46 46-222 08 70
Mobile: +46 767-64 52 66
E-mail: Eva.Friman [at] biol.lu.se
For inquiries about Bioinformatics contact:
Telephone: +46 46-222 94 19
E-mail: Bjorn.Canback [at] biol.lu.se
The facility acknowledges generous financial support during the years from the Knut and Alice Wallenberg Foundation (SWEGENE programme), the Crafoord Foundation and the Faculty of Science, Lund University.