Products & Solutions
FAQs
1. What are the sample requirements for sequencing?
Sample requirements depends on the sequencing application:
- Genomic DNA requiring nebulization: >5 µg of double-stranded DNA (dsDNA) in a 10 µL volume.
- DNA fragments 200 - 700 bp: >3 µg of dsDNA in a 10 µL volume.
- PCR amplicons that incorporate our fusion primers (<500 bp): ~100 ng of dsDNA in a 10 µL volume.
2. What quality of DNA is required?
A spectroscopic measurement of the A260/280 ratio of ~1.8 provides a good estimate of sample quality. We also request a 2% agarose gel image of the DNA sample(s) run with a 1 kb ladder to assess incoming sample quality in comparison to our internal QC.
3. Will you check the quality of my DNA before sequencing?
All incoming sample quantity is determined by fluorometry and quality is evaluated using either an Agilent bioanalyzer or agarose gel to assess the size distribution of the samples. If sample quantity or quality is not adequate, a replacement sample will be requested before proceeding with the project.
4. What method of quantification should I use?
Fluorometry provides the most reproducible and accurate quantification. Please note that many nano-scale fluorometers can dramatically under-estimate sample quantity which can delay projects if additional an sample must be requested for processing. We recommend using larger sample volumes for quantification.
5. What buffer should I suspend my samples in?
Samples should be submitted in a 10 µL volume of 1X TE buffer.
6. How should samples be shipped?
Click here for sample submission guidelines (PDF).
7. What is the accuracy of the sequences that I receive?
The GS-FLX provides a single-read accuracy of 99.5% or better for each individual read of 200-300 bp. This corresponds to roughly a Q40 quality score. This allows consensus accuracy of 99.99% with appropriate depth.
8. What coverage is needed for my genome sequencing project?
20-fold coverage of single-ended reads is recommended for de novo sequencing initiatives which will yield a high quality draft assembly. Incorporating Long-Tag Paired-End reads will aid in assembly and orient the scaffold the resulting contigs.
15-fold coverage is recommended for re-sequencing applications and mapping against a reference sequence to have high confidence in variant calling.
For Ultra-Deep sequencing applications, the depth of coverage is dependent upon application type and experimental goals.
9. What is an amplicon sequencing project?
Amplicon sequencing projects incorporate 19-mer 454 Fusion Primers into your gene-specific primers which allows them to proceed directly into emPCR for sequencing. This is often associated with Ultra-Deep sequencing applications. Please refer to our Guide to Amplicon Sequencing (link to Guide to Amplicon Sequencing App note) for additional information.
10. What are your fusion primer sequences?
Primer A: 5?-GCCTCCCTCGCGCCATCAG-3?
Primer B: 5?-GCCTTGCCAGCCCGCTCAG-3?
Roche has an exclusive partnership with Integrated DNA Technologies for design and creation of 454 Fusion Primers. Please visit www.idtdna.com for more information.
Or see our Guide to Amplicon Sequencing application note for more details on how to incorporate our A and B primers into your project.
11. How many samples can I analyze per sequencing run?
The number of samples analyzed in parallel depends upon the total size of the samples and the desired depth of coverage. Sequencing can be performed in one, two, four, eight, or sixteen sample formats based on gasket applied to the PicoTiter plate. Additionally 454's MIDs allow more flexibility for combining samples for parallel sequencing.
12. What are Multiplex Identifiers (MIDs)?
Our MID sequencing adaptors incorporate 10 bp tag sequences that are designed to take into account the instrument nucleotide flow order and ensure that >5 sequencing errors are required to misidentify a read. This allows multiple sequencing libraries to be pooled and the reads sorted by sample during subsequent bioinformatics analysis.
13. Can you help me design barcode tags for amplicon sequencing?
454 does not assist in sample barcode design however multiple published references develop tagging strategies similar to MIDs sequencing.
14. Can I use your MID sequences for my amplicon project?
Yes, please see below for a list of the MID sequences that can be incorporated into your fusion primers.
MID1 ACGAGTGCGT
MID2 ACGCTCGACA
MID3 AGACGCACTC
MID4 AGCACTGTAG
MID5 ATCAGACACG
MID6 ATATCGCGAG
MID7 CGTGTCTCTA
MID8 CTCGCGTGTC
MID9 TAGTATCAGC
MID10 TCTCTATGCG
MID11 TGATACGTCT
MID12 TACTGAGCTA
15. How long do projects take?
The length of a project depends on number of samples, genome size and data analysis requirements. The average turn around time for standard projects is 4-6 weeks from project start date, assuming there are no sample quality issues.
16. What is the difference between a draft and a finished genome?
There is no universal consensus on these definitions as is discussed in "What is Finished, and Why Does it Matter" Mardis et. al. Genome Research. Vol. 12, Issue 5, 669-671, May 2002. The definitions below are generalized but may not apply to all circumstances.
A finished sequence defines a highly-accurate genome assembly typically with less than 1 error for every 106 bases and contiguous sequences covering all reliably sequenced regions arranged in the appropriate order. The assembly has typically had gap closure via directed PCR and subsequent sequencing.
A draft genome assembly typically results from computational assembly of shotgun reads and generally has not incorporated biochemical confirmation of the resulting assembly.
The University of Maryland Center for Bioinformatics & Computational Biology has an excellent genome assembly primer covering the complexities of genome assembly.
17. Has anyone done a project like mine before?
The publications section of our website is an excellent resource for investigating previous projects completed using 454 Sequencing. The application notes specifically address a number of project types. Please contact us to discuss your project with our knowledgeable staff who can advise you in optimally applying 454 Sequencing to your project.