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imarkRoy J. CarverUniversity of Illinois
Biotechnology Center

High-Throughput Sequencing and Genotyping Unit
Director: Alvaro Hernandez Ph. D.
340 Edward R. Madigan Laboratory,
1201 W. Gregory Drive, Urbana, IL 61801
Phone: (217) 244-3480     
FAX: (217) 265-5066


Illumina HiSeq 4000 and 2500


Dr. Alvaro Hernandez, Director of DNA Services



8 lane HiSeq flowcell


2 flowcells on the HiSeq2500

Our facility offers a full range of services for library construction and sequencing with two HiSeq 4000 (a unit of the HiSeq x10) and two V4-capable HiSeq 2500.  For sequencing on the MiSeq platform, please visit the MiSeq page.

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 and now 10x Genomics Chromium libraries for denovo genome assembly, genome phasing and single-cell transcriptomes.


Turnaround time is typically one to three weeks from the day we receive the samples.

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.
Read lengths:  

50nt = small RNAs
100 to 250nt = DNAseq, RNAseq, ChIPseq, mate-pairs, bisulfite-treated, etc

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.
Typical Yields:

The HiSeq2500 in High Output mode uses an 8 lane flowcell and typically generates 180 to 250 million single-reads per lane with the V4 chemistry or 350 to 500 million paired-reads per lane. This is equivalent to 18 to 25 GBases of single 100nt reads or 35 to 50 GBases of 100nt paired-reads (reads can be 50nt, 100nt or 160nt in length).


The HiSeq2500 in Rapid Mode uses a 2 lane flowcell and generates 220-400 million paired-reads per lane (160nt or 250nt single reads).


The HiSeq 4000 yields 300 to 400 million single-reads and twice as many paired-reads per lane. Reads are up to 150nt in length.


Library construction:

Libraries in our facility are constructed with the TruSeq Sample Prep or Nextera kits from Illumina. All our RNAseq libraries are strand-specific.  

Libraries are quantitated with fluorometry (Qubit), run on a Bioanalyzer of Fragment Analyzer, diluted to 10nM and quantitated by 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 Nextera Mate-Pair Library kit.


TruSeq Synthetic Long Reads (formerly known as Moleculo) are here!

Sample submission requirements:

Sample Type

Minimum Quantity needed for library construction

Maximum Volume

Special instructions


Genomic DNA for shotgun libraries

10ng to 1μg

100μl 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

100ng to 1μg

20 to 50μl 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).



10μl in EB buffer

*DNA must be sonicated/fragmented to a size of 100-600bp (<600bp) before ChIP procedure.

* Quantitation with fluorometry (Qubit or Picogreen) is critical.
* Send gel picture or bioanalyzer trace if available.


Small RNA

1μg of total RNA
or 100ng of enriched small RNAs

20 to 50μl, 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.


Genomic DNA for 5kb, 8kb or 15kb mate-pairs libraries


100μl in EB buffer

Integrity of the DNA is critical for successful library construction. The majority of the DNA should be greater than 40kb in size when run on low percentage agarose gel. See images below.

Libraries can be made with a jumping size of ~5kb, ~ 8kb or ~ 15kb.

Customers submitting their own libraries:

Libraries must either be compatible with the read1, index and read 2 primers used by the current HiSeq chemistry or be submitted with at least 20ul of 100uM custom Read1, index and Read2 primers. Libraries should be submitted as 10nM dilutions if possible.

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
Once submitted, we will quantitate your pooled libraries 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.


28S and 18S rRNA bands are sharp. No smear below the 18S band. No genomic DNA is seen on the gel.


Description: Macintosh HD:Users:alvaro:Desktop:WebPage:bioanalyzer.jpg
High quality total RNA has RIN (RNA integrity number) of 8 or higher.

Description: Macintosh HD:Users:alvaro:Desktop:WebPage:PGFE.jpg

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 ( at 217-244-3480 or Chris Wright, Assistant Director of DNA Services ( 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.


All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.

Last edited: 2 Sept 2011