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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
Email: aghernan@illinois.edu 

SERVICES/EQUIPMENT

Illumina MiSeq V3 And MiSeq FGx Forensic Genomics System

MiSeq v3

Illumina MiSeq v3 and MiSeq FGx Services

We offer the full suite of MiSeq sequencing capabilities on our 3 in-house MiSeq platforms.  One is upgraded to the MiSeq FGx Forensic Genomics System, which runs all MiSeq kits as well as the Forensic FGx sequencing kit. The MiSeq FGx Forensic Genomics System is the first fully validated system for forensic genomics.  The FGx Forensic platform uses dedicated library preparation kits and customized instrument control software to simultaneously interrogate every locus currently in use in crime labs today plus hundreds more, including autosomal short tandem repeats (STRs), mitochondrial DNA, Y-STRs and single nucleotide polymorphisms (SNPs).  When combined with proprietary ForenSeq analytical software (Illumina), it supports reliable analysis of both routine and challenging forensic samples. You can read more about this amazing system here.

Guidelines for DNA Sample Submission for Illumina Sequencing Services

Our facility offers a full range of services for library construction and sequencing with the MiSeq v3 platform.  This platform generates 30-50 million 2x300 bp reads in a 3-day sequencing run, perfect for BAC/fosmid sequencing, sequencing of small genomes, metagenomic projects, denovo transcriptome assembly and many other applications.

We have extensive experience with the Illumina platforms and offer the fully upgraded 2x300 bp MiSeq v3 to our customers to complete their next-gen sequencing needs. We are running the newest, most robust MiSeq platform available.  With 5 HiSeq 2500 systems and 3 MiSeq, one of which is upgraded to the Forensic Genomics System (FGx), we have gained expertise over the years in Illumina library construction and sequencing and can easily apply this knowledge to the MiSeq platform.  We will be happy to help you meet your research needs using this platform alone or combined with data from our Illumina HiSeq2500s, or our traditional Sanger platforms.

Sequencing options (MiSeq):

Paired-reads: Library fragments are sequenced from both ends (paired-ends). Users can request smaller output 2x250nt Nano runs,or 2x250nt or 2x300nt bulk runs.
Library barcoding: All libraries are individually barcoded and can be multiplexed on a lane. The number of barcoded libraries that can be multiplexed per flowcell depends on the desired number of reads per library/sample.
Typical Yields:

The MiSeq v2 Nano uses a single lane flowcell and typically generates 1-2M paired-reads per lane.

The MiSeq v2 bulk run uses a single lane flowcell and typically generates 20-40M paired-reads per lane (amplicon output is ~10-20M reads).

The MiSeq v3 uses a single lane flowcell and typically generates 30 to 50 million paired-reads per lane. This is equivalent to 9 to 15GB of 300nt paired-reads. Amplicon output is ~20-40M reads.

 

Sequencing options (MiSeq FGx Forensic Genomics System):

Paired-reads: Library fragments are sequenced using an Illumina FGx sequencing kit for 350nt from one end and 30nt from the second end to fully sequence all target loci.
Library barcoding: All libraries are individually barcoded and can be multiplexed on a lane. For casework sample, Illumina recommends no more than 30 samples per pool.  For database samples, Illumina recommends no more than 96 samples per pool.
Typical Yields:

The MiSeq FGx sequencing run uses a single lane flowcell and typically generates 2-4M total analyzed reads after processing through the ForenSeq Universal Analysis sofware (Illumina).

Library construction:

Libraries in our facility are constructed with the TruSeq Sample Prep or Nextera kits from Illumina.  We also offer amplicon preparation from genomic DNA samples using the Fluidigm platform through our sister unit, Functional Genomics, with a wide variety of 16S, 18S, ITS, and other PCR primers available.  Please visit our Fluidigm 16S amplicon page, or contact us for more information on this system.

Libraries for the FGx system are prepared using the ForenSeq DNA Signature Prep kit from Illumina.  This kit can amplify over 200 loci covering autosomal STRs, X- and Y-STR loci and forensic SNPs along with additional phenotypic informative and ancestry informative SNPs, all sequenced and analyzed within a single run.

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 to ~15kb apart in the genome are paired-end sequenced, are constructed with the Nextera Mate Pair Library Prep kit.

Sample submission requirements:

Sample Type

 Quantity requested for library construction (total)

Maximum Volume

Special instructions

Options

Genomic DNA for shotgun libraries or FGx preparation

100ng - 1μg

 

FGx only: preps can be performed with 1ng of DNA. Please contact us for more details.

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

500ng - 1.5ug

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 poly-A mRNA (eukaryotic samples) or removal of ribosomal RNA (bacterial, metagenomic, human, other).

ChIP DNA

10ng-200ng

20μl, in EB buffer

* 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 small RNA

10μl, 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 3kb to 15kb mate-pairs libraries

20-40μg

100μl, 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 to ~ 15kb.

Customers submitting their own libraries:

Libraries must be compatible with the read1, index and read 2 primers used by the current MiSeq v3 chemistry or submitted with at least 15 ul of 100 uM read1, index, and read2 custom primers.  Users are highly encouraged to check with the sequencing lab before beginning construction of their own custom libraries (ie: 16S). 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.

Click here for more information on HiSeq2000/2500 and Gel/Bioanalyzer images of genomic DNA and Total RNA

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.

Pricing

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.