Illumina Sequencing Service
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Dr. Alvaro Hernandez, Director of DNA Services |
8 lane HiSeq flowcell |
2 flowcells on the HiSeq2000 |
Our facility offers a full range of services for library construction and sequencing with the Illumina HiSeq2000.
We have extensive experience in denovo genome and transcriptome sequencing, genome and transcriptome resequencing, profiling of gene expression,
comparative genomics and other applications using RNAseq, DNAseq, ChIPseq and small RNAs.
Sequencing options:
| Single-reads or paired-reads: |
Library fragments can be sequenced from one end (single-reads) or from both ends (paired-ends).
Single-reads are typically used for resequencing, gene expression profiling, ChIPseq, small RNA sequencing.
Paired-reads are most commonly used for denovo assembly and other applications.
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| Read lengths: |
35nt = small RNAs, mate-pair libraries
75nt or 100nt = DNAseq, RNAseq, ChIPseq
Can be customized up to a current maximum length of 100nt.
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| Library barcoding: |
All libraries are individually barcoded and can be multiplexed on a lane. The number of barcoded libraries
that can be multiplexed per lane depends on the desired number of reads per library/sample.
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| Typical Yields: |
The HiSeq2000 uses an 8 lane flowcell and typically generates 100 to 200 million single-reads per lane or
200 to 400 million paired-reads per lane. This is equivalent to 10 to 20GB of single 100nt reads or 20 to 40GB
of 100nt paired-reads. |
Library construction:
Libraries in our facility are constructed with the TruSeq Sample Prep kits from Illumina.
Libraries are then quantitated with fluorometry (Qubit), run on an Agilent bioanalyzer chip, diluted to 10nM and quantitated with qPCR.
Mate-pairs libraries, in which two fragments that were originally~3kb or ~8kb apart in the genome are paired-end sequenced, are constructed with the Mate Pair Library Prep kit v2.
Sample submission requirements:
Sample Type |
Minimum Quantity needed for library construction |
Maximum Volume |
Special instructions |
Options |
Genomic DNA for shotgun libraries |
1μg |
100ul, in EB or TE buffers |
* Quantitation with fluorometry (Qubit or Picogreen) is critical.
* DNA can also be quantitated on a 1% agarose gel next to a 1 Kb mass DNA ladder.
* Submit a picture with an aliquot of the DNA on a 1% gel next to a ladder before sending the sample.
* See below for examples of high quality DNA. |
Fragment sizes can be customized within a range, i.e. libraries can be constructed with average fragment sizes of 180bp, 500bp, etc. |
Total RNA |
1ug |
20ul of RNAse-free water |
*Should be treated with DNase.
*Run RNA either on a 1% agarose gel next to a DNA ladder or on a bioanalyzer RNA chip and submit picture or .pdf file.
See below for examples of high quality total RNA. |
Customer submits high-quality total RNA.
The first step of the library construction process involves selection of mRNA (eukaryotic samples) or removal of ribosomal RNA (bacterial and metagenomic). |
ChIP DNA |
10ng |
10ul, in EB buffer |
* Quantitation with fluorometry (Qubit or Picogreen) is critical.
* Send gel picture or bioanalyzer trace if available. |
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Small RNA |
1μg of total RNA
or 100ng of small RNA |
10ul, in RNAse-free water |
See instructions for Total RNA above. It is critical that total RNA is not purified with columns that remove fragments <100nt.
Kits that enrich for the small RNA fraction can also be used. |
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Genomic DNA for 3kb or 10kb mate-pairs libraries |
20-40μg |
100ul, in EB buffer |
Integrity of the DNA is critical for successful library construction. The majority of the DNA should be greater than 50kb in size when run on low percentage agarose gel. See images below. |
Libraries can be made with a jumping size of ~3kb or ~ 8kb. |
Customers submitting their own libraries:
Libraries must be compatible with the read1, index and read 2 primers used by the current HiSeq2000 chemistry. Libraries need to be submitted as 10nM dilutions.
For multiplexed libraries, please submit a final pool at 10nM (i.e. do not submit individual libraries before pooling). Dilutions to 10nM should be based on quantitation
by fluorometry and average size determined with a bioanalyzer chip. Enter these values in this attached calculation sheet|
For maximum yields we strongly recommend that 10nM dilutions are quantitated with qPCR.
Gel and Bioanalyzer images with genomic DNA and Total RNA:
Genomic DNA on a
1% agarose gel |
Total RNA on a
1% agarose gel |
Bioanalyzer trace of total RNA |
Genomic DNA for mate-pair libraries
on a Pulse Field Gel |
DNA in lanes 1, 3 and 4 is of high quality.
DNA in lane 2 shows signs of fragmentation/degradation.
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28S and 18S rRNA bands are sharp. No smear below the 18S band. No genomic DNA is seen on the gel.
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High quality total RNA has RIN (RNA integrity number) of 8 or higher.
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Lane 2: high quality DNA has large fragments >50kb.
Lane 3: partially degraded DNA has fragments <40kb. |
Please contact Dr. Alvaro Hernandez, Director of DNA Services (aghernan@illinois.edu)
at 217-244-3480 or
Chris Wright, Assistant Director of DNA Services (clwright@illinois.edu)
at 217-333-4372 to discuss ways the staff can be of assistance in
achieving your project goals or to receive a quote for your project.
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| Last edited: 2 Sept 2011 |
