Guidelines for DNA Sample Submission for
Roche/GS FLX+ Sequencing Services
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GS FLX+
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 Chris Wright, Assistant Director, DNA Services
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Titanium Shotgun
Sequencing |
Titanium Paired-End Sequencing |
Titanium Amplicon Sequencing |
Sample Type |
gDNA, plasmids, fosmids, BACs, long PCR products (>1.5 kb), RRLs, cDNA |
gDNA |
PCR products (amplicons)
Maximum recommended length 650 bps. |
DNA Quantity |
1 µg in maximum of 100 µl
TE or EB (1-3 ug for RRLs).
3ug is preferred.
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30 µg (20 kb PE), 15 µg (8 kb PE), or 5 µg (3 kb PE) in Tris-HCl or EB.
Do not useTE. |
20 ng in 10-20 µl TE or EB |
Calculated using fluorometry |
Calculated using fluorometry |
Calculated using fluorometry |
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 DNA Ladder2
*Fragments from RRLs should be 400-900 bps.
*cDNA must be free of long homopolymers. |
*gDNA must not be fragmented or degraded2. |
* Must contain A and B adaptors.
(LIB-L for bidirectional sequencing/LIG-L for unidirectional sequencing).
* Sample should be gel purified, followed by SPRI bead purification (Agencourt). |
Barcodes (MIDs) for sample multiplexing |
Over 100 available(12 available in-house). Sequencing is from one end (A adaptor) only. |
Optional. May significantly decrease true percent paired-end reads in final library. |
Over 100 available.
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Notes:
1) Qubit (Invitrogen) or similar method is recommended. Spectrophotometry, i.e. Nanodrop, is not compatible with this system.
2) 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
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Roche/454 GS FLX+ 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:
- Sample Specific library prep:
- Required for all samples that need fragmentation or that do notcontain Roche A and B adaptors.
- Involves shearing of DNA to fragments of 700-1100bp in length, ligation of adaptors for downstream amplification and sequencing; and final QC and quantitation of the prepared library.
- Barcodes (MIDs) can be added during library prep (up to 12 available in-house). The MID-tagged samples can be pooled for simultaneous amplification and sequencing (multiplexing).
- Titration run:
- Strongly recommended for all samples that will be run on 1/2 PTP or more.
- DNA libraries are amplified and sequenced at different concentrations to find the optimal conditions for sequencing on a full plate. Typically, 2-3 reactions are set up for each library and run on 1/16th lanes.
- Output from the titration run is very important for verifing library quality as well as proper MID distribution, and can be combined with bulk results. Typical output from a titration is ~25k reads per 1/16th region.
- Bulk sequencing run (Not including Amplicons):
- DNA from one library or from pooled barcoded libraries are sequenced on a PicoTiterPlate (PTP). Average fragment length for FLX+ is 400-600 bps. Plates can be divided in several regions as follows:
Regions: |
Number of reads (k) |
Number of bases (M) |
Full plate (PTP) |
900-1300 |
400-900 |
1/2 PTP |
450-650 |
200-450 |
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 be sequenced on the Titanium platform. Maximum recomended length is 650 nts. It is critical that PCR products are gel purified, followed by SPRI bead (Agencourt) purification for successful sequencing. Output for a Amplicon run are variable by nature and cannot be
guaranteed.
Please call the sequencing facility for the lastest information adaptor sequences before starting your amplicaon project!
Regions: |
Average Number of reads (k) |
Full plate (PTP) |
720-1040 |
1/2 PTP |
360-520 |
1/4th PTP |
100-250 |
1/8th PTP |
65-100 |
1/16th PTP |
20-30 |
Titanium Paired End (PE) Library Information
Titanium PE libraries are an extremely useful tool for de novo genome assembly.
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 3 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 PTPs
- 8 kb: 1 PTP
- 20 kb: ½ 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 an average length of ~135 bps 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 run output (see chart above) average read length is typical of Titanium chemistry (350-400bp).
GS FLX Titanium Chemistry Paired End
Minimum Library Expectations per Roche |
Span Size |
20kb |
8kb |
3kb |
HQ Linker Positive Reads
(of total HQ reads)
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≥
45 % |
≥ 45 % |
≥ 45 % |
| Average Tag Length |
≥ 135 bp |
≥ 135 bp |
≥ 135 bp |
Rate of Redundancy
(Per library)
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< 40% in up to 100K HQ reads |
< 40% in up to 250 K HQ reads |
< 40% in up to 500K
HQ reads |
True Pair Reads
(of Total HQ Reads) |
≥ 35% |
≥ 35% |
≥ 35% |
| Average Pair Distance |
16 - 24 kb |
6.4 - 9.6 kb |
2.4 - 3.6 kb |
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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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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
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| Last edited: 16 July 09 |
