For more detail on the steps involved in each application, please see the flow diagrams.
Here are slides to illustrate the sample preparation procedure that we do for each application. Hopefully these will give you a better understanding of what is involved in the preparation for each application. The only application that is not covered here is methylation, as the customer will be preparing their own sample for that method.
Genomic DNA Sample Preparation
Small RNA Sample
Preparation
mRNA Sequencing Sample Preparation
ChiP DNA Sample
Preparation
| Application | Minimum Input Requirements | Minimum Concentration | Buffer |
|---|---|---|---|
| Whole-genomic Sequencing | 5ug DNA | 500ng/ul | 1X TE |
| mRNA Sequencing | 20ug total RNA | 500ng/ul | DEPC water |
| ChIP Sequencing | 20ngDNA | 2ng/ul | 1X TE |
| Small RNA Analysis | 20ug total RNA | 500ng/ul | DEPC water |
| Methylation sequencing | Supply 50% of undiluted library preparation in EB buffer from QIAGEN purification | ||
| Mate Pair | 5-10ug | 500ng/ul | 1X TE |
Please refer below for more details on sample preparation requirements for each application.
The MGS supports single or paired-end reads for a wide range of genome sequencing applications.
For whole genome sequencing and re-sequencing, the MGS Illumina Sequencing Service provides customers with genomic DNA library preparation, cluster generation, and genomic analysis on the Illumina Genome Analyzer, with the option of single or paired-end reads. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
The MGS Illumina Sequencing Service requires customers to prepare their genomic DNA or cDNA amplicon to the following specifications:
Please refer to the list of prices for details on the cost of sample preparation, cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our whole genome sequencing and re-sequencing application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 36 bases or 50 bases. Also different applications can be run on one flow cell.
If mapping to a reference genome is required as part of an Illumina run, then the appropriate genome must be supplied at the time of sample submission. This can be either a genome of an organism as found in various sequence databases, or a "synthetic" genome where some preparatory work is required (see below). Whatever the source, the file format to supply is the standard FASTA format. Due to the potential size of such genomes, please supply your genome on either a CD or DVD.
ELAND -- the alignment program that is used as part of the pipeline -- has some limitations on the size of the genome that can be used. These limitations are purely computational, but they have to be adhered to. Quoting from the Illumina documentation:
The following requirements must be met:
- One file of 240x16 = 3824Mb
- 239 files, each of up to 16Mb in size
- Or something in between, e.g. 24 files of up to 160Mb each.
The NCBI human genome will fit. Please note that at this time (4th April 2007) ELAND does not check that the directory of "squashed" files it is matching against does not exceed these limits.
When a specific set of genes and/or sequences are to be aligned against -- for example EST libraries, highly fragmented genomes, and genomes that might come from a mixture of species -- we suggest you construct a "synthetic" genome and supply that as the reference sequence. This can be done by concatenating together all elements (genes, transcripts or other sequences), with adjacent elements separated by a stretch of Ns (ambiguous nucleotides). The purpose of the Ns is to ensure that Illumina reads cannot be split across two elements. We have found a "spacing" of 50 Ns works well. Once again, this data needs to be supplied in FASTA format, but as a single file.
Please remember that the alignment program (ELAND) will align the Illumina reads to the whole file, and the co-ordinates returned by the program will reflect this. It is therefore advisable to have two copies of your "synthetic" genome with the elements in the same order, one concatenated, and the other as a reference for subsequent analyses. There are a variety of ways to work out which element is bounded by a set of co-ordinates. This information is very important when looking at results through a genome viewer such as Gbrowse.
Small RNA discovery and analysis is a flexible platform on the Illumina Genome Analyzer, enabling the discovery and profiling of all forms of small non-coding RNA.
For small RNA analysis, the MGS Illumina Sequencing Service provides customers with sample preparation, cluster generation, and genomic analysis on the Illumina Genome Analyzer. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Please refer to the list of prices for details on the cost of sample preparation, cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our small RNA analysis application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina generated PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 36 bases or 50 bases. Also different applications can be run on one flow cell.
Chromatin immuno-precipiation analyses of protein-DNA interactions typically rely on DNA microarray hybridization quantification (Chip-chip), which is limited by the representation of the target genome on the DNA chip. The Illumina genome Analyzer system can be used for ChIP-Seq, where the immuno-precipitated DNA fragments are sequenced directly. This improves quantification and allows for whole-genome protein-DNA interactions to be surveyed and mapped to a sequenced genome with the one system.
For ChIP-Sequencing, the MGS Illumina Sequencing Service provides customers with sample preparation, cluster generation, and genomic analysis on the Illumina Genome Analyzer. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Customers must supply ChIP enriched, QPCR verified (PicoGreen Qualtified) DNA. The customer should supply a minimum of 20ng ChIP enriched DNA, at a minimum concentration of 2ng/ul in 1X TE buffer. Assaying of ChIP material by PCR must be carried out before delivery to our service facility. Our facility staff will elvaluate your results, and decide suitability for our subsequent preparation procedures. Please note that for Multiplex Indexing the same quantity of sample is required.
Please refer to the list of prices for details on the cost of sample preparation, cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our ChIP-Sequencing application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina generated PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 36 bases or 50 bases. Also different applications can be run on one flow cell.
mRNA-Sequencing can be used to obtain a more in-depth, comprehensive view of the transcriptome, revealing aspects previously unseen using array-based or expression sequence tag (EST) technologies. mRNA-Seq delivers unbiased and unparalleled information about the transcriptome because it does not require the designing of probes or primers. You can use this method to generate a full sequence from any poly-A tailed RNA to discover and profile novel transcripts, novel isoforms, alternative splice sites, rare transcripts, and cSNPs (coding region single nucleotide polymorphisms) in one experiment.
For mRNA-Sequencing, the MGS Illumina Sequencing Service provides customers with sample preparation, cluster generation, and genomic analysis on the Illumina Genome Analyzer. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Customers must supply a minimum of 20ug of highly purified total RNA at a minimum concentration of 500ng/ul in DEPC water. Please note that for Multiplex Indexing the same quantity of sample is required.
Please refer to the list of prices for details on the cost of sample preparation, cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our mRNA-Sequencing application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina generated PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 36 bases or 50 bases. Also different applications can be run on one flow cell.
DNA methylation plays a critical role in the regulation of gene expression and is an essential mechanism for normal cellular development. Methylation profiling is a valuable epigenetic tool for understanding the effects of aberrant methylation both in basic research and in clinical applications. Quantitative methylation measurement at the single-CpG-site level offers the highest resolution for understanding epigenetic changes. You can get a complete view of the epigenome at single-base resolution with the Illumina Genome Analyzer.
For Methylation Sequencing, the MGS Illumina Sequencing Service provides customers with cluster generation and genomic analysis on the Illumina Genome Analyzer. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Please note that it is the responsibility of the customer to do the sample preparation and give the sample to the MGS at a concentration of 10nM and supply a minimum volume of 10ul, ready for cluster generation on the flow cell. Please supply the Agilent 2100 Bioanalyzer report. In the absence of a bioanalyzer, please supply 50% of the undiluted PCR product. Please also note that methylation sequencing is not supported by Illumina. It can be very tricky to get high quality libraries and difficult to reproduce from tube to tube. If you do wish to proceed with doing any methylation sequencing on the Illumina through the MGS, it will be totally experimental, and the costs involved with sample preparation, cluster generation and GA analysis you will incur, whether you get data which meets your requirements or not.
Below is a list of the reagents which are needed for the sample preparation for Methylation Sequencing. Please note that it is the customer's responsibility to purchase the reagents for the sample preparation:
From Illumina Inc. you will have to purchase the following kits:
You can purchase the Illumina reagents above from Illumina Australia Pty Ltd. Their address and contact details are:
Illumina Australia Pty Ltd
3/1 International Court
Scoresby
Victoria 3179
Australia
Phone: +61 3 9212 9900
Fax: +61 3 9212 9901
E-mail: anzorders@illumina.com
Please refer to Prices for details on the cost of cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our Methylation Sequencing application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Below are two PDF documents for customers to download, giving information on the Paired-End sample preparation required for methylation sequencing, bisulfite sequence analysis FAQs, and Illumina notes for methylation sequencing. Within these documents there is reference to papers written on whole genome scale methylation sequencing on the Illumina platform.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina generated PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 35 bases, 50 bases or 75 bases. Also different applications can be run on one flow cell.
Agilent SureSelect Target Enrichment is useful when a researcher is only interested in sequencing a particular segment of the genome, just the translated part, for example. In this case, the Agilent SureSelect platform can be used to capture a subset of exons or other genome targets and wash away the rest of the genome prior to sequencing. SureSelect replaces other labor-intensive methods of targeted re-sequencing such as polymerase chain reaction (PCR) techniques that are a major bottleneck in most next-generation sequencing workflows.
The initial Agilent genome partitioning product is a ready-to-use kit containing a customer-specified mixture of up to 55,000 biotinylated RNA probes delivered in a single tube. The capture probes are 120 base pairs long, the longest currently on the market for this application. This makes them very effective at capturing DNA containing unknown mutations such as single nucleotide polymorphisms, insertions or deletions.
The MGS requires the customers to design their own custom SureSelect mixtures using the Agilent eArray online design tool, which contains many key genomes and also lets users upload their own sequences. Please refer to https://earray.chem.agilent.com, for more information. The customer must then provide the MGS with the SureSelect Oligo Capture Library (biotinylated RNA library – baits) and their purified genomic DNA, refer below.
Agilent SureSelect kits come in a range of sizes for 10s to 1000s of samples. For more information, please visit http://www.opengenomics.com/SureSelect.
The MGS Illumina Sequencing Service requires customers to prepare their genomic DNA or for SureSelect Targeted Sequencing to the following specifications:
Prices and online submission will be available soon.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina PhiX control sample. You are not required by the service to use all 7 channels of the flow cell, as we can combine different customer samples in one flow cell depending on the length of read you are requesting, 36 bases, 50 or 75 bases. Also different applications can be run on one flow cell.
For the Mate Pair Sequencing Assay, the MGS Illumina Sequencing Service provides customers with Mate Pair library preparation, cluster generation, and genomic analysis on the Illumina Genome Analyzer, with paired end reads only. Post pipeline analysis is also included, with the generation of either FASTA-A or FASTA-Q files with base-calling quality scores, for customer assembly. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
The MGS Illumina Sequencing Service requires customers to prepare their genomic DNA to the following specifications:
Please note that for Multiplex Indexing the same quantity of sample is required. If the customer does their own sample preparation please supply 50% of the undiluted library preparation in EB buffer from the QIAGEN purification.
Please refer to the list of prices for details on the cost of sample preparation, cluster generation, genomic analysis (generation and analysis of flow cell), and pipeline analysis for our Mate Pair Sequencing Assay application. Bioinformatics support and data assembly is available to customers on request, at an additional cost on a per project basis.
Our service can sequence up to 7 samples in a run, and the service uses the 8th channel for an Illumina PhiX control sample. You are not required by the service to use all 7 channels of the flow cell. It is recommended to run the Mate Pair Sequencing Assay application on a 35 base paired end run. Please refer to our section Mate Pair Sequencing Assay for details.
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