Guidelines for DNA Sample Submission for
Roche/454 Sequencing Services
|
Titanium Shotgun
Sequencing
|
Titanium Paired-End Sequencing |
Amplicon Sequencing (FLX only) |
Sample Type |
gDNA, plasmids, fosmids, BACs, long PCR products (>1.5 kb), RRLs, cDNA1 |
gDNA |
PCR products <800 bp |
DNA Quantity |
5 µg in maximum of 100 µl
TE or EB (1-3 ug for RRLs)
|
30 µg (20 kb PE), 15 µg (8 kb PE), or 5 µg (3 kb PE) in Tris-HC or EB.
Do not useTE. |
50 ng in maximum of 20 µl TE or EB |
Calculated using fluorometry2 |
Calculated using fluorometry2 |
Calculated using fluorometry2 |
DNA Quality and Features |
*gDNA should not be degraded.
Fragment size should be >1.5 kb as verified on 1% agarose gel with 1 Kb Mass Ladder3
*Fragments from RRLs should be 400-900 bps.
*cDNA must be free of long homopolymers1 |
*gDNA must not be fragmented or degraded3
|
* Must contain FLX A and B adaptors. Contact the facility for further information.
* Sample should be gel purified, followed by SPRI bead purification (Agencourt) |
Barcodes (MIDs) for sample multiplexing |
Up to 100 available. Sequencing is from one end (A adaptor) only. |
Same as Shotgun Library |
Up to 100 available. Sequencing can be from one or both ends. |
Notes:
1) Please visit our Library Services page for descriptions on cDNA synthesis and construction of custom
primary/normalized/subtracted cDNA libraries
2) Qubit (invitrogen) or similar method is recommended. Spectrophotometry, i.e. Nanodrop, is not compatible with this system.
3) Please send us an email with an image of your sample run on a 1% agarose gel before submitting your DNA for sequencing.
Examples of high-quality and low-quality gDNA samples are provided below.
High-quality gDNA on 1% Agarose Gel:
Lane 1: Ladder
Lane 2: gDNA Sample
|
Low-quality gDNA on 1% Agarose Gel:
Lane 1: Ladder
Lane 2: gDNA Sample
|
Roche/454 Technology and Sequencing Services
The Roche/454 GS FLX Sequencer utilizes Pyrosequencing technology to produce millions of high-quality DNA sequence reads from a single PTP (PicoTiterPlate) run. The process involves several steps:
- 454 library prep
- Required for all samples that need fragmentation or contain no 454 adaptors.
- Involves shearing of DNA to fragments to 500-800bp in length, ligation of adaptors for amplification and sequencing and conversion to a single-stranded template DNA library (sstDNA).
- Barcodes (MIDs) can be added during library prep (up to 100 available). The MID-tagged samples can be pooled for simultaneous amplification and sequencing (multiplexing).
- Titration run
- Required for all samples that will be run on 1 or more PTPs.
- sstDNA libraries are amplified and sequenced at different concentrations to find the optimal conditions for sequencing on a full plate. Typically, 3-4 reactions are set up for each library and run on a 1/16 th lane each.
- Output from titrations can also be used to verify library quality and proper MID distribution, and can be combined with bulk results. Typical output from a titration is ~25k reads per 1/16 th region.
- Bulk sequencing run
- sstDNA from one library or from pooled barcoded sstDNA libraries are sequenced on a PicoTiterPlate (PTP). Average fragment length is 350-400 bps. Plates can be divided in several regions as follows:
Regions: |
Number of reads (k) |
Number of bases (M) |
Full plate (PTP) |
900-1300 |
360-560 |
1/2 PTP |
450-650 |
180-280 |
1/4th PTP |
160-250 |
60-110 |
1/8th PTP |
80-120 |
30-55 |
1/16th PTP |
25-40 |
10-20 |
PCR Product (Amplicons) Information
PCR products (amplicons) smaller than 1kb in length can currently only be sequenced on the FLX platform, which yields fragments with an average length of 220bps. Kits for the Titanium version are expected by Fall 2009. It is critical that PCR products are gel purified, followed by SPRI bead (Agencourt) purification for successful sequencing.
Regions: |
Average Number of reads (k) |
Average Number of bases (M) |
Full plate (PTP) |
300-400 |
70-100 |
1/2 PTP |
150-200 |
35-50 |
1/4th PTP |
60-70 |
14-17 |
1/8th PTP |
30 |
5-7 |
1/16th PTP |
9-12 |
2-3 |
Titanium Paired End (PE) Library Information
Titanium PE libraries are an extremely useful tool for de novo genome assembly. Currently, we offer 3 kb, 8 kb, and 20 kb Paired End libraries. Barcoded adaptors (MIDs) may be utilized for PE libraries. Please see our Sample Submission guide for specifics on sample requirements.
General recommendations for choosing which PE library is right for your de novo sequencing project:
- Smaller (>5 Mb) microbial genomes: a single 8 kb PE library may generate enough data for assembly.
- Larger/more complex microbial genomes: 15x coverage using a mix of shotgun, 3kb or 8 kb PE libraries.
- More complex genomes: 12x shotgun, 3x 3 kb PE, 2x 20 kb PE, and possibly 2x 8 kb PE libraries.
Other factors to consider:
- Multiple PE libraries of the same size span may need to be constructed depending upon your coverage depth required due to the limited number of unique fragments present in each PE library. Maximum recommended level of sequencing per PE library before oversampling is reached is as follows:
- 3 kb: 2 bulk PTPs
- 8 kb: 1 bulk PTP
- 20 kb: ½ bulk PTP
- Although the DNA sample requirement is large, especially for 20 kb PE libraries, multiple libraries can often be made from the same original sample. Library preparation pricing is discounted in this situation.
- Due to the nature of PE library construction, you should expect ~135 bps of sequence on each side of the linker, redundancy of <40% in up to 100k high quality reads, and a minimum of 35% true paired-end reads from all 3 PE library types. Total output from PE runs should mimic gDNA Titanium run output (see chart above).
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.
|
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
|
| Last edited: 16 July 09 |
