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Sample Submission Requirements:

Our facility offers a full range of services for library construction and sequencing with the NovaSeq 6000, MiSeq, Oxford Nanopore GridION and the 10x Genomics Chromium. The HiSeq 4000 and 2500 have been decommissioned.

We have extensive experience in denovo genome and transcriptome sequencing, genome and transcriptome resequencing, profiling of gene expression, comparative genomics and other applications using RNA-Seq, DNA_Seq, ChIP-Seq, bisulfite-treatment DNA, and small RNAs, 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, ChIP-Seq, and small RNA sequencing. Paired-reads are most commonly used for de novo assembly, characterization of splice variants, and other applications that benefit from more information from the sequenced fragments.

Typical Read Lengths and Yields:

NovaSeq 6000:  

  • SP flowcell:
    • Single-reads 100bp or paired-reads 2x150bp or 2x250bp.
    • 400 to 500 million single reads per lane.
    • 800+ million paired reads per lane.
  • S1 flowcell:
    •  Single-reads 100bp, paired reads 2x100bp or 2x150bp.
    • 800+ million single-reads.
    • 1.5 billion paired-reads per lane.
  • S2 flowcell:
    •  Single-reads 100bp.
    • 1.5 billion single-reads per lane.
  • S4 flowcell:
    •  Paired reads 2x150bp.
    • 5-6 billion reads per lane.

MiSeq:

  • V3 bulk run:
    • 10-30 million paired-reads
    • Paired reads up to 300nt in length available.
  • V2 bulk run:
    • 6-20 million paired-reads
    • Paired reads up to 250nt in length available.
  • V2 Nano run:
    • 500k-2 million paired-reads
    • Paired reads up to 250nt in length available.

HiSeq 4000 (legacy):

  • 8 lane flowcell:
    • 300-400 million single-reads per lane.
    • 650-800 million paired-reads per lane.
    • Up to 120Gbases per lane.
    • Single and paired-read options from 50nt to 150nt available.

HiSeq 2500 (legacy):

  • 2 lane Rapid V2 flowcell:
    • 150 - 200 million single-reads per lane.
    • 220 - 400 million paired-reads per lane.
    • Single and paired-read options from 50nt to 260nt available.

Library construction:

Libraries in our facility are constructed with kits from Illumina using UNIQUE DUAL INDEXES (UDI's) to avoid index switching. Our RNAseq libraries are strand-specific.  

All constructed libraries are quantitated with fluorometry (Qubit), checked for proper size distribution and complete primer/adaptor removal on a Fragment Analyzer or Bioanalyzer, diluted to 5nM, and quantitated by qPCR prior to sequencing.

Sample submission requirements:

Sample Type

Minimum Quantity needed for library construction

Volume

Special instructions

Options

Genomic DNA for shotgun libraries

10ng to 1μg

20-50μl in 10mM Tris

* 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.

* DNA should be clean of contaminants, salts, etc

Fragment sizes can be customized to a tight size or wide range, i.e. libraries can be constructed with average fragment sizes of 180bp, 500bp, 400-800bp, etc.

Total RNA

50ng to 1μg

20 to 50μl of RNAse-free water

*RNA must 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 free of contaminating DNA.
The first step of the library construction process involves selection of mRNA (eukaryotic samples) or removal of ribosomal RNA (bacterial, metagenomic or eukaryotic).

ChIP DNA

5ng-10+ng

10μl in 10mM Tris buffer

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

* Quantitation with fluorometry (Qubit or Picogreen) is critical.

* Send gel image or bioanalyzer trace of sonicated input DNA 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 2-15kb mate-pairs libraries

5-10μg

100μl in 10mM Tris 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.

*Do not submit DNA in buffer with EDTA.

Libraries are typically made with a jump size of ~2-4kb, ~ 8kb or 10-12kb but can also be customized for your project.

Genomic DNA for 10x Genomics genome assembly/phasing 1μg of HMW DNA or DNA embedded in agarose plugs 50-100μl or agarose plugs *DNA must be in fragments > 50kb, ideally > 100kb. The presence of fragments < 40kb negatively impact the results of the assembly. Required sequencing is 1 lane of HiSeq 4000 2x150 per GB of genome.
Single-Cell RNA-Seq (10x Genomics) 500k -1M cells at least 20μl

*Elimination of dead cells is critical, viability should be >70%. Cells can be processed through a Dead Cell removal kit if required.

*Concentration should be at least 5000 cells/ul (5M/ml).

*Starting cell suspension will be counted (live/dead measure included), washed twice in PBS with 0.04% BSA, and counted again to ensure proper cell capture.

Each library will be made from 500 to 10,000 cells, depending upon your project need.

Required sequencing is a minimum of 50,000 2x100nt paired-reads per cell; 100k reads/cell and 2x150nt length reads are recommended.

Genomic DNA for Oxford-Nanopore

1-2μg

500ng for rapid libraries

 20-100μl in 10mM Tris buffer DNA must be free of proteins, polysaccharides and polyphenols and any other contaminants. The A260/280 and A260/A230 ratios should be 1.8 or higher libraries can be made with an average fragment length of 8-15kb (higher output) or with longer fragments.
RNA for direct RNA sequencing on Oxford-Nanopore at least 500ng of polyA+ RNA 2--100μl of water If 500ng of polyA+ RNA are not available, mRNA can be converted to cDNA and PCR-amplified  

Customers submitting their own libraries:

Libraries must either be compatible with the read1, index and read 2 primers used by the current NovaSeq/HiSeq/MiSeq chemistry or be submitted with at least 20ul of 100uM custom Read1, index and Read2 primers. Libraries should be submitted pooled and at 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/fragment analyzer. Enter these values in this attached calculation sheet

Once submitted, we will quantitate your pooled libraries with qPCR prior to sequencing.

Gel and Bioanalyzer images of 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 10x Genomics or mate-pair libraries
on a Pulse Field Gel

Genomic DNA
DNA in lanes 1, 3 and 4 is of high quality. DNA in lane 2 shows signs of fragmentation/degradation.

 

Total RNA
28S and 18S rRNA bands are sharp. No smear below the 18S band. No genomic DNA is seen on the gel.
Bioanalyzer
High quality total RNA has RIN (RNA integrity number) of 8 or higher.

 

PGFE
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, submission forms, grant support or other information needed.

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.