SERVICES CATALOGUE WITH SUBMISSION GUIDELINES

SERVICES CATALOGUE WITH SUBMISSION GUIDELINES 3921 Montgomery Road Cincinnati, Ohio 45212 513-841-2428 www.agctsequencing.com

CONTENTS Welcome Dye Terminator Sequencing DNA Sequencing Services - Full Service Sequencing - Load Only Sequencing - Heterozygote Sequencing - Template Scanning - Concentration Adjustment - Gene Assembly - Troubleshooting Services - Universal Primers Available Fragment Analysis Services - Full Service Fragment Analysis - Load Only Fragment Analysis - Fragment bp Size Capability - Recommended Fluorescent Dyes - Special Projects Appendix A: Troubleshooting Guide - Appearance of Good Data - Template Concentration Issues - Double Sequence Results - Salts and Other Contaminants - Sudden Stops - Poly Base Regions - Nicked DNA Appendix B: Single Colony Isolation Protocol Appendix C: Primer Design Appendix D: Agarose Gel Electrophoresis Contact Information Page 1 Page 2 Page 3 Page 3 Page 3 Page 4 Page 4 Page 5 Page 5 Page 6 Page 8 Page 9 Page 10 Page 11 Page 11 Page 11 Page 12 Page 13 Page 13 Page 14 Page 15 Page 16 Page 17 Page 19 Page 20 Page 22 Page 23 Page 25 Page 26

Welcome to DNA Analysis DNA analysis was founded in 2008 with the purpose of bringing the academic core lab environment to a commercial facility. Our focus is on providing you with good service. The company founders have more than 20 years combined experience providing genetic services to academic researchers. What separates our company from the large companies is that you can contact and speak directly with laboratory personnel working with your samples. We establish a strong professional relationship with every research submitting samples. We are easy to contact by phone or email ready to consult and provide you with assistance. Our company provides quality DNA sequencing, fragment analysis and related services. Each result is carefully reviewed for quality and accuracy before we release the results to you. This allows us to become more familiar with the samples you submit and any special chemistries required for difficult regions. When results are complete, they will be uploaded to your free secure account ready for you to download. Submitted samples are generally stored for a minimum of 3 months where they can be used for future testing. Samples and primers can be placed in long term storage at no additional cost by request. For additional information please check our website at www.agctsequending.com. Or simply contact us by phone (513) 841-2428 or email (info.agctsequencing.com). Page 1

Dye Terminator Sequencing DNA Analysis uses dye terminator chemistry (Sanger Sequencing) for automated sequencing applications. Dye terminator chemistry is relatively similar to basic PCR. A template and primer are combined in a reaction mix containing dntps, buffer and Taq polymerase. The differences with basic PCR amplification is that a single primer is used and amplification is linear from the original template. Dye terminators also include terminating nucleotides, or ddntps. The ddntps will randomly terminate PCR extension on some of the products creating a ladder that increase in length by one base. Each product is separated as it migrates through the capillary matrix during electrophoresis to produce the sequence. Page 2

1. Full Service Sequencing Service To submit samples for full service sequencing, template DNA and primers should be submitted in separate tubes. However, if the same template is sequenced using more than 1 primer, you will NOT need to submit a separate tube the template. Similarly, you will only need to submit a single tube of primer to be used for multiple templates. Please submit template and primer concentrations as provided to prevent additional charges for concentration adjustments. Plasmid < 6 kb 50 ng/ ul 10 ul per reaction Plasmid > 6 kb 75 ng/ ul 10 ul per reaction PCR products 1 ng per 100 bp 10 ul per reaction PCR products 10 ng per kb 10 ul per reaction BACs and PACs please contact us before submission Once your samples have been received at our facility, our technical staff will - Combine the template, primer and premix to perform dyeterminator amplification. - Purify the post PCR samples. - Load onto the capillary genetic analyzer. - Review, analyze and edit the final sequence result before uploading into your secure online account. 2. Load Only Sequencing To submit samples for load only sequencing service, you will perform dye terminator amplification using the Life Technologies Big Dye Terminator V3.1 Cycle Sequencing Kit (No. 4336917) and purify the samples before submission. This is our most cost effective sequencing service. However, it does limit our ability to troubleshot. Once the samples are received at our facility, our technical staff will - Re-suspend the samples in high-deionized formamide. - Load the samples on the capillary genetic analyzer. - Review, analyze and edit the final sequence result before uploading into your secure online account. Every sample submitted to DNA Analysis will be reviewed and edited to correct for software generated base miscalls. Not only do we correct base miscalls during review, we also become more familiar with your samples for future submissions. Page 3

3. Heterozygote Sequencing Heterozygote sequencing uses the dye terminator chemistry to detect single nucleotide polymorphisms, or SNPs. Accurate detection of a polymorphic base requires relatively equal signal for each base. Although once requiring dye primer chemistry and universal primers, dye terminator version 3.1 allows any primer to be used to detect polymorphic bases. A polymorphism is generally detected when two separate bases occupy the same base position. Because two bases are present in one position, the signal strength for both bases is half when compared to non-polymorphic bases as shown. There are no special requirements for submitting samples for heterozygote sequencing. However, please note that you are looking for polymorphic bases under special instructions so we include this during review and editing. 4. Template Scanning Scanning spectroscopy is an indicator of both template concentration and quality. A typical test scan of DNA measures absorbance using Ultra Violet light in wavelengths ranging between 220 nm and 350 nm. Quality DNA produces a Gaussian curve with the maximum absorbance at 260 nm and a 260 nm/280 nm ratio value of approximately 1.8. A secondary peak is also produced at the lowest wavelength as an indicator of salts and possible contaminants. DNA Analysis utilizes the Nanodrop spectrometer to scan DNA templates. Nanodrop technology provides and accurate scan measurement and requires only 1 ul of the template. Page 4

Nanodrop scan showing Gaussian curve for samples of different concentration 5. Concentration Adjustment Dye terminator sequencing is sensitive to the concentration of the template DNA including plasmid preparations and PCR products. Not only can DNA Analysis scan the templates submitted for concentration, we will also adjust the concentration to improve the quality of DNA sequencing for a small fee. Often, concentration adjustments has led to improved sequencing results. 6. Gene Assembly We can complete the entire sequence for your larger DNA inserts or amplified PCR product using basic primer walking protocols. This could help free up more time for your other laboratory work. Simply submit the template and beginning primers so we can generate the initial set of sequence results. Following completed analysis and editing of the results we will continue to design the primers and primer walk through the remainder of the sequence. Page 5

We will assemble and review sequence results using the electropherogram. This provides a two step editing process. First sequence results are edited before assembly and then again after assembly to provide the most accurate consensus sequence. It is important to note that average turn-around time required to produce each set of primers and generate the next set of sequences is 2 to 3 days. Final results of the assembly will be provided in an assembly report including the primers, assembled sequence results and consensus. 7. Troubleshooting Sequence quality is dependent on many factors including quality of the template and primers, presence of GC rich regions, salts, formation of hairpins and if the template is nicked. While most laboratories may provide free reloads to confirm a failed sequence, this does not help determine the cause for why some samples fail to provide results. DNA Analysis incorporates a set of protocols and guidelines to try and determine why some samples fail to sequence. This includes review of the entire set of results, Nanodrop scanning and agarose gel electrophoresis. Combined, these tests generally determine the whether the template contains some inhibitors that prevent successfully sequencing. Page 6

A general review of results could show a pattern in the entire group of samples. For example, a group of submitted templates may work, but fail with the same primer. This would indicate the problem is related to the quality of the primer or the annealing site. Nanodrop scanning will allow us to assess the quality of the template and primer. Often the problem is the result of insufficient concentration or presence of salts. Nanodrop technology does not allow us to determine if the template is nicked. Nicked DNA changes the conformation of the template DNA and prevents Taq polymerase from forming a proper fit necessary for amplification. Nicked DNA is best determined using agarose gel electrophoresis. GC rich regions and secondary structure that form hairpins can also prevent extension of the dye terminator amplification. Often, these sequence related issues can be determined during the review of the sequencing result. DNA Analysis makes every effort to determine the cause for problematic samples and correct the problem before reloading or reworking the sample. Page 7

8. Universal Primers Provided by DNA Analysis There are no cost to you when selecting our universal primers for sequencing M13(-21) For M13(-40) M13Rev T7 T3 SP6 T7 Term CMV F BGH R pgex5' pgex3' GL1 GL2 RV3 RV4 KS SK T25V A25B TGT AAA ACG ACG GCC AGT GTT TTC CCA GTC ACG AC CAG GAA ACA GCT ATG ACC TAA TAC GAC TCA CTA TAG GG ATT AAC CCT CAC TAA AGG GA TAT TTA GGT GAC ACT ATA G TAT GCT AGT TAT TGC TCA G CAA GCG GCC TCT GAT AAC CA TAG AAG GCA CAG TCG AGG GGG CTG GCA AGC CAC GTT TGG TG CCG GGA GCT GCA TGT GTC AGA GG TGT ATC TTA TGG TAC TGT AAC TG CTT TAT GTT TTT GGC GTC TTC CA CTA GCA AAA TAG GCT GTC CC GAC GAT AGT CAT GCC CCG CG TCG AGG TCG ACG GTA TC CGC TCT AGA ACT AGT GGA TC (T)25V where V = A, G and C (A)25B where B = G, T and C Page 8

Fragment Analysis Fragment analysis is a method used to detect variance in base pair size between PCR fragments generated from multiple samples. It is less stringent and sensitive than automated sequencing and more cost effective. A designated loci is amplified by PCR using forward and reverse primer that mark the area of interest. However, to use an automated platform, the forward primer is tagged with a fluorophore for detection purposes. Agarose gel electrophoresis has been used previously for similar fragment analysis applications. But, the detection of different fragment sizes is fairly limiting. Automated fragment analysis applications is highly reproducible and can detect differences between fragments of one base pair. Page 9

DNA Analysis provides both full service fragment analysis applications and more cost effective load only services. A second advantage of automated fragment analysis is that different markers can be combined together in a single test capillary by using fluorophores with different emission spectra which appear as different colors. In fact, the automated system has the capability to combine up to 16 markers in a single capillary allowing tremendous multiplexing capability. 1. Full Service Fragment Analysis Applications To provide full service fragment analysis, you would submit the template DNA along with forward and reverse markers for each loci. DNA Analysis would provide the following steps to complete analysis. New projects generally require some optimization time to design the best method of PCR to amplify the products. For smaller projects we do not recommend mutiplexing directly in the PCR. - Amplify products by PCR using the markers provided. It should be noted that multiplexing can occur either directly in the PCR or samples could be combined following PCR. For larger sample groups, it is worthwhile to try and multiplex markers directly in the PCR. Smaller groups of samples are recommended to combine samples after PCR. In general, PCR optimization is the most complex step especially when mutiple markers are combined in a single reaction. - Purify amplified products in preparation for loading on the Capillary genetic analyzer. - Combine samples with GS standard before loading on the genetic analyzer. - Perform electrophoresis. - Analyze the fragment analysis data and review results. - Upload completed results into your free and secure online account. Results are reviewed by qualified technicians and summarized using Applied Biosystems Genotyper software. We provide you with the raw data files, pdf of the Genotyper comparing each sample and tabular results in an Excel file. Please let us know if you would like to view the results in a specific format and we will try to accommodate you. Page 10

2. Cost Effective Load Only Fragment Analysis DNA Analysis provides load only services for laboratories that prefer to amplify products in their laboratories and submit samples ready to load on the genetic analyzer. This is a popular service for most fragment analysis services. Once the samples are received our technicians will - Combine samples with GS standard before loading on the genetic analyzer. - Perform electrophoresis. - Analyze the fragment analysis data and review results. - Upload completed results into your free and secure online account. 3. Fragment Size Limitations DNA Analysis uses two separate size standards. GS500 and GS2500. The GS500 standard is used to detect fragments in the range 100 bp to 450 bp. The GS2500 provides a more broad array of fragment sizes that can be tested. However, GS2500 is less accurate than GS500 because of reduced linearity of the standard curve. The technical staff at DNA Analysis is experienced using both standards to provide the most accurate results for your fragment analysis data. 4. Recommended Fluorescent Dye Standards The genetic analyzer requires calibration for different dye standards. We recommend for your fragment applications the following calibrated standards for your fluorophore. 1. 5-FAM, NED and HEX 2. 6 FAM NED and HEX Our GS standard for both GS500 and GS2500 use ROX as the fluorophore. Therefore, this fluorophore should not be used for your samples. The fluorophore for the standard should always be unique from the samples. Page 11

5. Special Project Needs Fragment analysis applications cover a broad spectrum of test procedures. We have designed and analyzed projects that include microsatellite analysis, RFLP, AFLP and metagenomic studies. Each project is different and requires special design in order to complete. We recommend that you contact us for your project needs before submitting samples. We will be able to discuss the necessary design parameters and provide an estimate of the steps and time necessary to complete the project efficiently while producing quality results. Page 12

Appendix A: Sequence Troubleshooting Guide 1. Interpretation of Good Data Raw Data The raw data can be visualized from the main sequence result file. The software to review the raw data is Data Analysis. It is used by the automated capillary sequencer for data analysis. First we look at the baselines. They should run flat across the window. The peaks in the raw data should have good height and should run across the window. If excess terminators and/or strong dye blobs are present the peaks on the raw data are automatically re-scaled by the software. They may look small, but still yield useable data. Check the start point from the raw data - the automatic analysis often mis-assigns the start point. If there are big peaks at the start of the lane (left hand side of the raw data window), the analysis should be reset to start after these peaks. Otherwise, the analyzed data will re-scale all of the data to include these peaks. This will make the rest of the peaks appear smaller. PCR products need to be cut alter the stop point. This will re-scale the peaks making the analyzed data easier to interpret. Page 13

2. Issues Related to Template Concentration Dye Terminator Sanger sequencing chemistry is sensitive to the concentration of the template. To optimize sequence data quality, it is important that you follow recommended guidelines for template concentrations. High Concentration Plasmid DNA with excessive amount of template generally causes depletion of the dntps similar to the effects in PCR and also depletion of the ddntps. Typically, the raw data for excessive template concentration is as follows. A simple solutions for solving high plasmid concentration issues is to re-quantify the DNA and adjust concentration before reworking the sample. Excessive concentration in smaller PCR fragments could produce a different result as shown above because the smaller product does not necessarily deplete dntps and ddntps. But, excessive loading of the sample on a capillary genetic analyzer causes slow or sluggish migration during electrophoresis. The raw data for excessive PCR product concentration is shown. Page 14

As the product proceeds along the capillary, resolution is lost early making base calling impossible. For excessive concentration of PCR products, a simple resolution is to dilute the final product and reload on the capillary genetic analyzer. Low Concentration Plasmid DNA and PCR products with low template concentration often produce low signal strengths when viewing original electropherograms and for the raw data. Slightly low concentrations may have little effect in the final result. However, excessively low concentrations may fail to produce any result. 3. Results Showing Double Sequence A result that shows a double sequence could be related to template contamination or possibly a secondary priming site on the template submitted. The results for both conditions is usually different and helps to identify the cause of double sequence. Plasmid contamination generally appears as clean sequence up until the point of insert in direction of one primer. The reverse sequence often appears as clean sequence through the insert. The figure shows a typical result when plasmid DNA has secondary template contamination. Page 15

It should be noted that the double sequence is marked both by presence of 2 bases at each base location as well as a drop in the peak height. To prevent double sequence as the result of template contamination we recommend single colony isolation in Appendix B. Double priming is differentiated from template contamination because the double sequence result is present from the beginning of the sequence as shown in the figure. Both priming sites will generate sequence beginning with the smallest bases number and the secondary sequence generated will overlay on top of the primary sequence. 4. Salts and Other Contaminants Samples contaminated with salts can produce results similar to low concentration by increasing the amount of background included in the result. Page 16

Even though the sample may produce sufficient signal strength, the background is also amplified in the overall signal. This could result in an early loss of resolution and shorter read length. 5. Sudden Stops in the Sequence Sudden stops in sequence data can often result from a number of conditions found in the sequence of the template. GC rich regions, secondary structure, poly G regions and GT repeat regions can all cause a sudden stop in the sequence. Most problematic templates that result in sudden stops can be resolved by using a specialized or more robust chemistry, or by sequencing in the reverse direction. Results showing a loss in resolution from GC rich regions or poly GT areas are shown. The first sequence shows the result using basic dye terminator chemistry compared to the result using more robust dye terminator chemistry. Page 17

GT repeat regions show results similar to GC rich problematic areas. Dye terminator chemistry designed for GT areas generally works well to sequence through this difficult region. One of the more difficult regions to resolve using dye terminator chemistry is the hairpin loop caused by complimentary regions present on the same strand. This can produce one of several types of results and is often difficult to diagnose during analysis. Most often, the sequence comes to a sudden stop as if PCR extension reached an extreme poly G region. However, GC bases may or may not be present. A partial hairpin can also form when some of the sequence amplification extended beyond the hairpin while other templates terminated. This could appear as a double sequence. Or, a region of the inserted template could be absent in one or both directions. Page 18

A variety of special chemistries and PCR conditions have been developed to sequence through the more difficult secondary regions and success of these protocols has been relatively high. DNA Analysis will review the region where the sudden stop occurs in attempt to determine the most likely cause. GC rich regions and poly GT areas are generally easy to identify. Hairpins resulting from complimentary regions are more difficult. 6. Poly Base Regions A very common result when using cdna as the template is stuttering caused by Taq polymerase slippage. This occurs when the polymerase enzyme slips along poly T areas from the poly A tail generated in cdna. The result is appearance of stuttered bases directly following the poly T area of the sequence. Stuttering can also occur for poly A areas. Despite this problematic condition, stuttering is easily resolved using primers that anneal directly to the poly T or poly A areas on the template. Page 19

8. Nicked DNA Nicked plasmid DNA will cause sequence failure. The reason for sequence failure is because the nick in the DNA loosens the DNA strand and prevents Taq polymerase from forming the proper enzyme lock along the template. As a result, the enzyme is unable to initiate extension during dye terminator PCR. Scanning the template spectrophotometrically does not provide the means to detect a nicked template. However, agarose gel electrophoresis would show whether the template is nicked because nicked DNA migrates more slowly along the gel than supercoiled DNA. Most plasmid preparations using a kit will result in both supercoiled DNA and nicked DNA being present in the sample. This is very common. However, presence of only nicked DNA will result in sequencing failure. Page 20

Nicked DNA Supercoiled DNA Generally nicked DNA occurs from physical breakage of the DNA during the purification process. It is recommended that you follow the instructions included with the kit especially when vortexing is involved. Running samples on an agarose gel will show whether nicked DNA is present before samples are submitted for sequencing. DNA Analysis uses several technics to determine whether a template is nicked including agarose gel electrophoresis and special universal primers that sequence along the ampicillin or kanamycin gene present in nearly all plasmids. Page 21

Appendix B: Single Colony Isolation Protocol Plasmid preparations begin with isolation of a single colony from a plate followed by growth of the colony in liquid media prior to preparation. However, a single colony does not necessarily grow from a single isolated bacteria. Selecting a colony that grows from a group of bacteria could cause template contamination in the DNA template resulting in double sequence as shown in the Troubleshooting guide 3. Therefore, we recommend a single colony isolation. Single Isolation is an additional step where the selected colony taken from the first plate, is then spread on a second plate. This brings a level of assurance that the colony selected for the template preparation is homogenous and prevents the possibility of template contamination. Page 22

Appendix C: Guidelines for Selecting Sequencing Primers 1. Primer length should be in the range of 18 to 22 bases. Primers less than 18 bases will have a low melting temperature (Tm values) and might not anneal to the template. There is some flexibility for designing primers longer than 18 bases. Longer primers are frequently designed from template regions that are AT-rich and need additional bases to increase the Tm value. 2. The primer should have GC content of 50% to 55%. This is the equivalent of 9 or 10 GC bases included in an 18 base primer. Sometimes there are regions on a template that are AT-rich which prevents meeting this guideline. In those cases it is recommended to design a primer longer than 18 bases. 3. Primers should have a GC-lock on the 3 end. A GC-lock is designed when 2 of the final 3 bases is a G or a C. The 3 base should always be a G or a C. 4. The melting temperature of any good primer should be in the range of 50 O C to 55 O C. However, guidelines particularly related to Tm value have some flexibility. Melting temperatures are directly related to the PCR cycle annealing temperature. Tm values that are too low may not anneal well during PCR. High values could be too stringent causing difficulty locating the correct annealing site on the template. 5. The primer should not include poly base regions. This is when 4 or more bases in a row are the same. This guideline helps prevent potential slippage in which the primer shifts from the annealed position. 6. Four or more bases that compliment either direction of the primer should be avoided. This prevents the primer from annealing to itself and forming what is referred to as primer-dimer. Primer-dimers have the capability of amplifying the primer itself causing short secondary sequence. Page 23

PCR Specific Guidelines 7. Forward and reverse primers used in PCR amplification should have similar melting temperatures (+/- 2 O C). This allows a 4 O C difference in total melting temperatures. Researchers involved in using PCR amplification will use primer Tm values in an effort to optimize PCR cycles. Similar Tm values for forward and reverse primers aid optimization efforts. Multiplex PCR applications using multiple primer pairs should all have similar Tm values. A wide range in primer melting temperature complicates PCR optimization. 8. Forward and reverse primers should not have regions 4 bases or longer that compliment. Just like a primer used in Sanger sequencing, forward and reverse primers used in PCR can anneal to each other and form primer-dimers. 9. The Tm values for tailed primers should include the tail in calculating melting temperature. Yes, melting temperatures will be greater than 55 O C. However, the additional bases in the tail will add to the amplified PCR fragment and become part of the priming site. Tailed primers are often used to add restriction sites to an amplified product. Page 24

Appendix D: Recommendations for Agarose Gel Electrophoresis The correct percent agarose gel is dependent on the size of the fragment that will be tested. Plasmid DNA preparations that are 5 kb to7 kb resolve well on a 1% gel. Large PCR fragments that are similar in size to plasmid DNA could also resolve on a 1 % percent gel. However, small PCR fragments that require smaller pore size for better resolution require a higher percent gel. General guidelines for mixing the correct percent gel are provided in table 1. Page 25

CONTACTS Doug Bintzler - Laboratory Director Doug.bintzler@agctsequencing.com Doug was the Director of the DNA Core Facility at the University of Cincinnati College of Medicine. Under the direction of Faculty Advisor, Dr. Joanna Groden, Doug initiated the automated sequencing and fragment analysis programs and continued to develop the automated DNA synthesis service at the university. During his 15 years at the university, Doug became experienced in using slab gel automated systems and capillary systems. He improved techniques for working with problematic templates including GC rich and secondary related problems. In 1995, Doug joined an international organization called the Association of Biomolecular Resource Facilities (ABRF). In 1997 he became a more active member by joining the Fragment Analysis Research Group (FARG), one of the many research committees within the ABRF. He chaired FARG from 1999 to 2002. In 2006 he joined the DNA Sequencing Research Group (DSRG) and continues to be an active member. He has published works on developing DNA sequencing techniques and on DNA synthesis. He has been an invited speaker on topics that include DNA synthesis, DNA sequencing and fragment analysis. Michael Jordan - Sequencing Director Michael.jordan@agctsequencing.com Michael had 10 years of experience working in the University of Cincinnati DNA Core Facility before partnering with Doug Bintzler to found DNA Analysis, LLC in 2008. During this time he learned both automated DNA sequencing and automated DNA synthesis techniques. He became skilled in providing a high degree of quality in these services while maintaining an excellent level of productivity. Michael learned sequencing using slab gels originally. This taught him precision in his techniques as the procedure is not forgiving. When capillary sequencing was introduced at the DNA Core he learned to hone his skills to maximize his efficiency. Through years of experience he acquired the ability to identify problematic templates and still produce quality results. This ability to troubleshoot difficult areas of sequencing is one of the cornerstones of DNA Analysis, LLC. An area of expertise for Michael is gene assembly. Over the years he has developed the skills to design primers that, with the proper custom chemistries, can sequence through extremely difficult regions of DNA. His abilities in this area have even led to authorship in a publication. DNA Analysis, LLC www.agctsequencing.com 513-841-2428 3921 Montgomery Road Cincinnati, Ohio 45212 Page 26

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