• Overview – Sequencing vs. FA
• DNA Sequencing: Service Levels
• Fragment Analysis: Service Levels
• Sample Preparation – General
• Physical Submission of Samples
• Online Submission Request
• Suggestions
• Turnaround Time
• Data Analysis
The 3130xl Genetic Analyzers (Applied Biosystems) can run a wide variety of sequencing and fragment analysis applications – including microsatellite analysis, AFLP, LOH, SNP validation, and SNP screening – as well as de novo sequencing and resequencing (mutational profiling). Typically, our two 3130xl’s are set up for standard DNA Sanger-Sequencing (≤900 bp) with POP7 polymer and 50-cm arrays, a combination which also performs well for fragment analysis; upon special request, we can install 80-cm arrays (≥1000 bp reads) or 36-cm arrays (rapid sequencing and fragment analysis). Requests for sample processing and data retrieval are both handled online through the Genomics Core website.
Overview – Sequencing vs. FA
DNA Sequencing sample preparation: Samples will not be processed unless they were properly prepared as noted below.
– 'Electrophoresis-Only' option: Samples must be cleaned in a manner that removes the unincorporated BigDye terminators (via ethanol
precipitation or commercial methods). Samples must then be dried and resuspended (typically in 15 μl of Hi-Di formamide; however, other
formats may be accommodated upon special request) prior to submittal to the Genomics Core.
– Template option: As described at the Full-Service DNA Sequencing Instructions webpage, clients typically must clean and aliquot
the required volume of DNA for each reaction into the tubes (or 96-well plate). In some cases, clients may request that the
Genomics Core perform Template cleanup and quantitation; however, this requires a concurrent submission
request for additional service (see EtOH-EDTA Purification and
DNA Column Purification & Size Selection).
DNA Fragment Analysis sample preparation: Samples will not be processed unless they were properly prepared as noted below.
– 'Electrophoresis-Only' option: Samples are submitted as completed reactions, including the addition of size standards, in 10-15 µl of HiDi Formamide. Samples should be prepared in accordance with the notes and example document shown below.
– Raw FA products option: As noted below, clients may request that the Core process raw FA products. In this case, pre-consultation is required to determine exactly what services the Core will provide and to establish the specific submission protocols for your project.
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Service Levels: DNA Sanger-Sequencing
▶
Electrophoresis-Only: Client performs BigDye sequencing reaction, cleans products, and
resuspends them in (preferably) ABI Hi-Di formamide... ready for electrophoresis
on the 3130xl.
Guidelines and requirements for DNA sequencing are specified in Sequencing Instructions.docx; see also Sequencing Secrets Revealed.docx. Additional assistance can be found in the Science Aid Center
and Documents & Protocols (under the heading of "ABI 3130xl Genetic Analyzers").
Samples must be sequenced with ABI BigDye
Terminator v3.1; otherwise, your samples may generate inaccurate base calls due to incompatibility with our
Spectral calibrations.
▶
DNA Templates: After consultation with Core staff, client submits DNA templates (see
Full-Service DNA Sequencing Instructions), with the Genomics Core performing the BigDye sequencing reactions and cleanup procedures, followed by electrophoresis on the 3130xl.
'Template' option requires prior approval by the Genomics Core to ensure client fully understands submission requirements.
Options exist for cleanup of the templates prior to performing the sequencing reactions; incurs additional reimbursement charges.
Reduced Core reimbursement rates are available for Bulk Template Submissions (i.e., ≥47 templates).
— Primers: Client supplies primers either in separate tubes (if submission meets the "Master-mix" requirements for primers) or pre-aliquoted into sequencing plate/tubes. The Core provides certain common primers free-of-charge.
— Difficult to Sequence (DTS) samples: Most samples will perform well using the standard cycling parameters and 0.5 ul of BigDye per reaction. However, if there are reasons to suspect that your samples will be very difficult to sequence (e.g., strong secondary structure, multiple homopolymers, microsatellites), you may opt for the DTS cycling parameters and additional BigDye per reaction. Alternatively, if you want to avoid the cost of extra BigDye, you may request just the DTS parameters or even your own cycling specfications. Please contact the Core for further details.
— Templates: Fastest, lowest-cost results are obtained if the client cleans & aliquots templates into sequencing plate/tubes with required DNA mass for each sequencing reaction. All wells must have same volume; if needed, dilute higher concentration templates in nuclease-free water or TVLE (both available at the 'Reagents & Supplies' link below). Alternatively, clients may submit 'raw' PCR products for cleaning and aliquoting by the Core; as described below, this option requires 1 or 2 additional online 'Service' requests:
A) If 1 well = DNA for 1 reaction:
– 1) DNA purification;
B) If 1 well = DNA for multiple reactions:
– 1) DNA purification; and,
– 2) Aliquoting cleaned templates into sequencing plate. ↵ Back to Top
Service Levels: DNA Fragment Analysis
— Submission Options
Fully prepared samples: Typically, samples are submitted as completed reactions, including the addition of size standards. As such, they require only sample denaturation (if desired) and electrophoresis on the ABI 3130xl.
Partially prepared samples: Clients may submit raw PCR products (i.e., FA products) and request that the Core dilute/aliquot the FA products and add the size-standard to all wells... prior to performing electrophoresis on the ABI 3130xl.
'Partially-prepared' option incurs additional reimbursement charges and requires prior approval by the Genomics Core to ensure client fully understands submission requirements.
Sample Denaturation: Upon request, FA samples will be heated and snap-cooled immediately prior to loading them on the 3130xl. When requesting this option, specify the denaturation temperature and duration in the 'Client Memo' field of your submission.
— Dye Sets
Before performing your reactions, verify with us that your dye set (see DyeSets.docx) is compatible with our Spectral calibrations
If not, either you will need to use a different dye set or we will need to order a dye matrix calibration kit to generate the required Spectral.
— Size Standards
The Core stocks LIZ 500 (Thermofisher Cat. No. 4322682); other size-standards can be ordered upon request.
Before choosing a size standard, please consult the "Sample Preparation" document shown below.
— Sample Preparation
Determining the correct sample dilution, amount of diluted sample input, & amount of size-standard is an iterative process. Please see FA_sample_preparation.xlsx for a guide to performing this process.
HiDi Formamide (Thermofisher Cat. No. 4311320) is required for both dilution of FA products and for the Size-Standard Master-mix (other formamides might not be properly buffered).
Water is not recommended for dilutions – mixing water with formamide generates formic acid ions, resulting in poor data quality.
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Sample Preparation – General
Reagents: We stock a variety of reagents related to DNA sequencing and fragment analysis (view
listing at Reagents & Supplies).
All requests must be made online (Log In) through the Genomics Core website.
Positive controls: including ≥1 p-ctrl (up to 3-5% of total samples) is highly recommended.
Sample Layout: The 3130xl injects samples in batches of 16 (i.e., 2 columns X 8 rows
per injection), and injections always start on 'odd-numbered' columns. Thus, your samples should be arranged in the
same manner to maximize the lifespan of the capillary arrays.
Sample Clean-up: The last step in generating high quality samples for DNA Sequencing is the
clean-up phase. In addition to removing
contaminates (such as salts, which affect sample injection and electrophoresis), it is crucial to completely
remove the unincorporated dye terminators.
Otherwise, those products will generate massive peaks ~70 bp into your sequence read, both obliterating ~20-bp of
data and squashing all subsequent peaks due to scaling of the electropherogram to the highest sample peak...
see also Lies, Damn Lies, and 'Seq-Files').
Sodium Acetate vs. EDTA: Do not
use sodium acetate to clean PCR products for DNA sequencing. Sodium acetate will tend to recover the original primers along with the PCR products, leading to poor sequencing results.
Blank wells: Ideally, 'blanks' should be placed at the end of your last Strip-tube or in the
last occupied column of your 96-well plate. Further, either do NOT add fluids to those blank wells or else clearly mark them as 'blanks' (e.g., with a thick 'slash' or an 'X').
Short DNA Fragments?: Rapid-Response DNA Sequencing is possible if you consolidate samples of similar desired read length into batches of 16. For example, if the maximum read length required is 200 bp for samples in A1-H2 and 800 bp for samples in A3-H4,
we can set the run module of A1-H2 for ~1/4 the time of the run module for A3-H4.
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Online Submission Request
Log In to the website, fill out the 'Request Form'
page, and submit your request. If you make an error in your request or upload the wrong Sample sheet,
you may revise that request if you do so before we begin processing it.
When your samples are loaded on the 3130xl, you will receive an automated email; you will
receive another automated email when your results are uploaded to the website (click on
'Submission History' and download the 'File'). If you do not receive the second automated email within 1-2 business days following the initial email, please contact the Genomics Core.
Files will be “zipped”, reducing ~30 Mb of data from a full plate (96 samples) to ~10 Mb.
Files will remain in your 'Submission History' for ≥90 days; if you need a copy of the data after it's purged, we can usually retrieve it from our archives.
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Physical Submission of Samples
Plates or Tubes?: Full details
are available at the Science Aid Center. Briefly, strip tubes can be loaded directly into the 3130xl's
96-well base plates, without having to transfer the samples to a 96-well plate. Thus, either format is
acceptable, provided you use plates that are known to fit on the 3130xl's 96-well plate bases or else
0.2 ml Strip tubes. However, samples
submitted in any other type of tube (i.e., >0.2 ml or individual tubes) will be rejected
due to the increased risk of loading errors, contamination, and sample loss.
Label Submission: Upon successful submission of your online request, the 'Submission #" will appear on your screen. Label the plate itself (or 0.2-ml tube rack) with: Submission #; and, Submitter's name (PI Name and Date, optional). Notations written on top of a sealing film
will be lost upon processing, so ensure that the labels are on the actual plate (or rack). Further, when
submitting tubes, you must clearly label the ends of each Strip-tube set (e.g., "T-1 and T-8").
Delivery Site: Transfer tubes to a Genomics Core rack (taking your own rack back to
your lab), and put samples in the Mini-fridge (by sink) in the Genomics Core (A628 Life Science Annex).
[Note: If the Core is locked, put your samples in the 'After-Hours' Mini-fridge located in the hallway (by Room A649).]
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Suggestions
Sequencing Problems?: Review
Science Aid Center.
Sample Clean-up: For DNA Sequencing reactions, ethanol-EDTA precipitation
is your cheapest option and can give excellent results (on par with or exceeding commercial kits)
when done correctly. Typically, Dye Terminators are fully removed when samples are spun upside-down to remove the ethanol solution; alternatively,
performing two ethanol washes and aspirating the solution can give adequate results. However, 'dumping & 'blotting' tubes leaves large amounts of dye terminators and gives inferior sequencing results.
Sealing plates: Either commercial sealing films or clear 3M® packing tape may be used. Further, blue "painters" tape has been successfully used to seal DNA Sequencing plates, and caps are always acceptable.
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Turnaround Time
'Electrophoresis-Only' option: Barring an unusual situation, results are available no later
than within 3 working days of submission. In fact, if samples are submitted before ~2:00 p.m.,
results are typically uploaded sometime the following business day – although more time is
required whenever an instrument must provide the maximum sequence length for a total of 192 samples
(i.e., 2 full plates).
'Raw Template' options: Additional time is required for performing the pre-3130xl activities needed with either raw DNA templates or raw FA products; however, unless other time-intensive activities
are in progress, 'Template' samples are usually on the 3130xl within 1-2 days of their initial submission... although it might be 2-3 days for submissions that need template clean-up prior to sequencing.
The processed data are then usually uploaded to the website sometime the following working day.
Standard run module: For DNA Sequencing, it takes ~2 hr for a single run (16 samples), ~6 hr for a half-plate (48 samples), and ~12 hr for a full plate (96 samples); faster times are possible when shorter sequence reads are requested (see 'Sample Preparation – General' above). For DNA FA samples, each run (16 samples) typically takes ~1 hour.
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Data Analysis
Properly analyzing Sanger-Sequencing data requires some expertise and training. Further, there are some aspects of the data that can be adjusted only by software on the instrument. Thus, if you have any concerns about your data, feel free to request a consultation!
Lies, Damn Lies, and 'Seq-Files': Sequence text files lie; electropherograms lie somewhat less!
- The text 'seq-file' created by the basecalling program makes no distinction between low-quality
and high-quality data. Thus, portions of the 'seq-file' will be inaccurate... especially
near the end of the read.
- Electrophoretic traces are scaled to the highest intensity of the sample — such that low-intensity
samples will show 'nice' peaks even for low-quality data (sometimes, even for 'noise'). The presence of
unincorporated dye terminators or fragment analysis primers exacerbates this effect.
- Therefore, trust in your data should come only through first checking the signal intensity with
Sequence Scanner, and then by comparing the seq-file to the electrophoretic trace file.
Further details and examples can be seen at
Why analyze data with Sequence Scanner? in the Science Aid Center.
Result formats: Results are available as text files and electropherograms. Text files can be opened in any text editor; electropherograms are accessible with freeware such as:
-
Sequence Scanner from ABI – provides access to raw data, signal intensity, and more;
- Connect— Cloud-based freeware (ThermoFisher).
- BioEdit - full-featured
program, including sequence alignment; and,
- Chromas LITE - basic
sequence manipulation.
Available software: The following ABI software packages are installed on the Core's Client PC Workstation:
• GeneMapper v5
• MicrobeBridge
• MVF (Minor Variant Finder
• PeakScanner v2
• Primer Express, v3.0.1
• Protein Thermal Shift (PTS), v1.4
• QuantStudio·6 qPCR, v1.3
• Sequencing Analysis v6
• Sequence Scanner, v2
• SeqScape Software v3
• Variant Reporter v2
— GeneMapper v5: A genotyping software package that provides DNA sizing and quality allele calls. This software specializes in multi-application functionality, including amplified fragment length polymorphism (AFLP®), loss of heterozygosity (LOH), microsatellite and SNP genotyping analysis. The software uses Process Quality Values (PQVs) for automated identification that reduces data review time for high throughput genotyping.
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— MicrobeBridge: This software connects *.ab1 data files (i.e., Sanger sequencing files) with the CDC’s MicrobeNet database for bacterial identification using 16S rRNA gene sequencing analysis. If using MicrobeNet directly, researchers must perform manual quality checks of sequence data assembly, manual examination of the assembled sequence, and a manual alignment search against Genbank to identify the species. MicrobeBridge automates the process by performing contig assembly, contig editing, primer trimming, and data quality checks of the imported *.ab1 data files; it also copies the contig sequence and provides a one-click connection to MicrobeNet.
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— MVF (Minor Variant Finder): This software permits calling low-frequency somatic variants in Sanger sequencing data; it can also be an important confirmatory method for next-generation sequencing (NGS) results. Using a control sample, the software's algorithm neutralizes background noise, enabling calling minor variants (≥5%).
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— PeakScanner v2: This is a freeware version of GeneMapper, which can be used to perform DNA fragment analysis. The software allows you to view, edit, analyze, print, and export fragment analysis data (generated using the ABI 3130xl Genetic Analyzer).
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— Primer Express, v3.0.1: This software can be used to design your own primers and probes using TaqMan and SYBR Green dye chemistries for real-time PCR applications. This software was developed specifically for use with Thermofisher's real-time PCR systems (e.g., QuantStudio·6).
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— Protein Thermal Shift (PTS), v1.4: This software was developed to analyze protein melt fluorescent readings directly from Applied Biosystems real-time PCR instrument files (e.g., QuantStudio·6). The software generates one or multiple melting temperature values (Tm) from QuantStudio·6 run files by two completely unrelated methods: the Boltzmann-derived Tm; and, the Derivative Curve-determined Tm. The two-state Boltzmann model normalizes across noisy undulations in the signal. However, those undulations might not be noise in the case of multi-domain proteins where they may correspond to different domains coming apart in stages. In those cases, the derivative method can show temperatures at which the local peaks occur. If the two-state model is a great fit for the data, the results should be in close agreement.
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— QuantStudio·6 qPCR, v1.3: This software allows the user to set up real-time Run files as well as open and analyze experiments generated with the QuantStudio·6 Real-Time PCR System.
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— Sequencing Analysis v6: Software enables you to basecall, trim, display, edit, and print data from the 3130xl platform for data analysis and quality control. Normally, this software is only used by the Genomics Core itself; however, for special situations, clients may wish to reanalyze their sequence data by tweaking certain basecalling parameters in the software.
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— Sequence Scanner, v2: This software provides six windows for analyzing Sanger sequencing trace data, allowing you to view, edit, print, and export data generated by the ABI 3130xl Genetic Analyzer (post processing by Sequencing Analysis software). Most critically, SequenceScanner allows you to evaluate the actual signal strength of your reactions through access to the original raw data. With minimal training, you can also use the software to troubleshoot poor sequencing reactions and to make judgements about the likelihood that the 3130 XL sequencer malfunctioned during your runs.
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— SeqScape Software v3: A resequencing package designed for mutation detection and analysis, SNP discovery and validation, pathogen sub-typing, allele identification, and sequence confirmation. It provides library functions for comparison to a known group of sequences, as well as 21 CFR Part 11 functionalities.
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— Variant Reporter v2: Performs comparative sequencing, also known as direct sequencing, medical sequencing, PCR sequencing, and resequencing with DNA sequencing files. The software is designed for reference-based and non-reference-based analysis such as mutation detection and analysis, SNP discovery and validation, and sequence confirmation. The algorithms will call SNPs, mutations, insertions, deletions, and heterozygous insertions⁄deletions.
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