Seminar: Sanger Sequencing

Transcription

1 Seminar: Sanger Sequencing 04. August 2010 Dr. René Gödde Produktspezialist Laborautomation

2 Seminar: Sanger Sequencing Beckman Coulter (Genomics) Agencourt CleanSEQ kit Requirements for a good sequencing result Troubleshooting Sanger sequencing

3 Beckman Coulter (Genomics)

4 Worldwide Organization HQ: Orange County, CA 10,000+ employees 250,000 placements in 130 countries Dedicated to biomedical testing

5 SIMPLIFY AUTOMATE INNOVATE

6 Agencourt History Founders developed SPRI nucleic acid purification chemistry at Whitehead which was used to sequence over 1/3 rd of the Human Genome, 1998 Agencourt Bioscience incorporated, July 2000 Began offering Sequencing Services, April 2001 Began offering NA purification kits, June 2003 Acquired by Beckman Coulter, June 2005 Acquisition of Cogenics, April 2009 Renamed to Beckman Coulter Genomics, August 2009 Today the world s largest commercial provider for genomic services and nucleic acid purification products!

7 Beckman Coulter Genomics Genomic Services Regulated Services DNA Sequencing NextGen Sequencing Gene Expression Genotyping Biologis Testing Nucleic Acid Extraction Biorepository Sample Submission Service Facilities Products SPRI Laboratory Automation DNA Purification and Cleanup Accessories Software SPRIworks Fragment Library System I & II SPRI-TE Nucleic Acid Extractor

8 Proven Experience Mammalian Gene Collection (MGC) cdna library screening Identified full length clones for all human/mouse genes Submitted over 1,250,000 sequences to GenBank Tetraodon nigroviridis Genome Project Sequenced > 1 billion high quality bases Collaboration with the Whitehead Institute Mus musculus Genome Project Sequenced > 1 billion shotgun sequencing bases 180,000 fosmid clones end sequenced Human Genome Project Generated > 70 Mb of draft sequence Finished > 46 of Chromosome 10 BACs Rattus norvegicus Genome Project Sequenced >3 billion high-quality bases Performed disease gene analysis Collaboration with the Baylor HGSC Methanobacterium Genome Project Finished 1.75 Mb genome sequence Detailed comparative analysis and annotation Ciona savignya Genome Project Sequenced 160,000 fosmid clones Collaboration with the Whitehead Institute Clostridium Genome Project Finished 4.1 Mb genome sequence Detailed comparative analysis and annotation Cancer Genome Anatomy Project (CGAP) SAGE sequencing Determined gene expression profiles for > 20 distinct tumor types Rickettsia sibirica Genome Project Created high and low copy genome libraries Sequenced complete genome to 12X coverage Initiated protein-protein interaction studies Felis catus Genome Project Sequenced >4 billion high-quality bases Whole genome assembly of 5 strains Chrysosporium lucknowense Genome Project Sequenced to 6X coverage and assembled Utilized by Dyadic for strain improvement

9 The Biomek Continuum: Flexible and Upgradeable Automation Assay Workstation Integrated System Process Workstation Liquid Handler

10 Automated dye removal at the CCG Biomek FXp DualHybrid Target Prep Express

11 Agencourt CleanSEQ kit

12 SPRI Technology SPRI - Solid Phase Reverse Immobilization Patented nucleic acid isolation process - Developed by Whitehead and exclusively licensed to Agencourt Paramagnetic bead-based - Uses Seradyn s Sera-Mag microparticles Reversible immobilization of DNA on bead surface Based on buffer conditions - nucleic acids bound in presence of proprietary buffer - no chaotrophic salts required - DNA eluted in aqueous solution

13 How Does SPRI Work? COO - - COO - COO -

14 How Does SPRI Work? COO - H 2 O - H 2 O H 2 O H 2 O COO - COO - EtOH H 2 O EtOH EtOH H 2 O H 2 O H 2 O

15 Typical SPRI Protocol Step 1: Nucleic Acid Immobilization Step 2: Contaminant Removal & Wash Step 3: Nucleic Acid Elution

16 CleanSEQ Competitive Quality pgem sequenced using M13 primers and 1/8th dilution BigDye v3.0 and sequenced on an ABI PRISM 3700 Sequencer

17 CleanSEQ vs. Ethanol x ~1,500 CleanSEQ reads ~1,500 EtOH reads Percentage of Reads Lower % Failure Lower Standard Deviation Higher Average Phred20 Phred 20 Read Length

18 Requirements for a good sequencing result

19 How does Sanger sequencing work?

20 What s inside a sequencing reaction? Primer * dntps * Water Template DNA * PCR Plasmide Fosmide/Cosmide BAC Additives DMSO Biotine BDT buffer Buffer salts * Sequenase * ddntps * It s all about balance! * specific for each reaction * contained within BigDye kit

21 Component requirements: Template DNA Concentration - Gel - Nanodrop - Picogreen - Bioanalyzer Purity - proper purification methods Specifity - no multiple templates Primer Concentration - dilute lyophilized primers properly Additives HPLC purified - non-hplc may lead to n-1 artifacts Design - use Primer 3 software or comparable Concentrations Only use when necessary (except DMSO)

22 Typical reaction setups: ABI recommended Agencourt recommended BigDye 8 µl 0.25 to 0.5 µl (1-2 µl for large/difficult templates) Template DNA 50 ng to 1 µg 50 ng to 1 µg Primer 1.6 to 3.2 pmol 1.6 to 5 pmol (probably 10 pmol for BACs) Water d.s. d.s. BDT buffer - 1x Total 20 µl 10 µl

23 Considerations when diluting BigDye When diluting BigDye, you re also diluting enzyme, salts, dntps and ddntps! May lead to disbalance in sequencing reaction! Fix by adding sequencing buffer and/or dntps! (see CCG web page for recipes)

24 Typical reaction setups: ABI recommended Agencourt recommended BigDye 8 µl 0.25 to 0.5 µl (1-2 µl for large/difficult templates) Template DNA 50 ng to 1 µg 50 ng to 1 µg Primer 1.6 to 3.2 pmol 1.6 to 5 pmol (probably 10 pmol for BACs) Water d.s. d.s. BDT buffer - 1x Total 20 µl 10 µl

25 Regarding template amounts Concentration [ng/µl] Number of molecules [mol]

26 Why? 100ng/µl of 0.5 kb PCR product 100ng/µl of 7 kb PCR product

27 Problem: [mol] hard to determine [mol] is sometimes hard to determine and prone to calculation failures Fix by using concentration with following formula: ng Template = 100ng x No. of kilobases Example: 0.5 kb PCR product 100ng x 0.5 = 50ng of DNA template per rxn

28 Typical reaction setups: ABI recommended Agencourt recommended BigDye 8 µl 0.25 to 0.5 µl (1-2 µl for large/difficult templates) Template DNA 50 ng to 1 µg 50 ng to 1 µg Primer 1.6 to 3.2 pmol 1.6 to 5 pmol (probably 10 pmol for BACs) Water d.s. d.s. BDT buffer - 1x Total 20 µl 10 µl

29 Troubleshooting Sanger sequencing

30 Ideas and tools Identify sequencing problems by looking at raw data, not analyzed data! Use trace viewer such as FinchTV! Use internet resources to indetify problems! In 99% of all instances the problem is IN the sequencing reaction!

31 Failed DNA sequencing reaction: How to identify The trace chromatogram has noisy or messy sequence peaks with low quality scores. No or little signal in the raw data channels except for leftover dye at the beginning of the trace. Signal strength in the raw channel usually below 100. The trace sequence does not match either the expected sequence or other sequences in GenBank.

32 Failed DNA sequencing reaction: Traces Figure 1. Example of a failed reaction trace. (A) Processed channel data. (B) Raw channel data at the same base location.

33 Failed DNA sequencing reaction: Causes Poor quality DNA. Very common when sequencing plasmid miniprep templates. Loss of the reaction during clean up. This can be a particular problem when using ethanol precipitation clean up protocols. Too much template DNA. Excess template can kill the sequencing reaction. Wrong primer used. More common than you might think! Bad water. The water used contains a sequencing inhibitor. Degraded or failed synthesis primer. Oligonucleotide synthesis is chemically complex and primer synthesis failures are fairly common. Dead sequencing chemistry. Can occur if the BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the Taq DNA polymerase or dye labeled nucleotides can be degraded. Blocked capillary. Every trace using a particular capillary fails. Can be identified by tracking trace quality on a trace by trace basis.

34 Failed DNA sequencing reaction: Troubleshooting Poor quality DNA. The best way of avoiding this problem is to not sequence plasmid DNA and sequence a PCR amplified fragment of the plasmid insert. If this is not possible then it is recommended that a plasmid miniprep kit is used. One tip is to perform a final ethanol precipitation on the kit purified plasmid DNA. This often solves problems with the quality of the template. Dirty cleanups like ExoSAP digestions may also cause purity problems. Loss of sequencing reaction during clean up. This can be avoided by not using an ethanol precipitation protocol to clean up the sequencing reaction. Too much template DNA was used. This can be avoid by checking the concentration of the template before sequencing. This may also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration! Wrong primer. This is simple to solve, but can be difficult to detect. Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" forward and reverse primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions (especially those of your unreliable lab colleagues!) and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.

35 Failed DNA sequencing reaction: Troubleshooting Bad water. Inhibitors can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap. Degraded primer. Don't use old diluted primer stocks. Store the primers in 10mM Tris/ 0.1 mm EDTA (ph 8.5) rather than water. Don't use other peoples stocks. If you have any doubt about how the primer quality is discard it and make up a fresh working solution from the primer stock. Failed oligonucleotide synthesis. If you suspect that the primer is poor quality check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems. Dead sequencing chemistry. This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles. Blocked capillary. Can be identified by tracking trace quality on a trace by trace basis. While this can be done manually we recommend using our QualTrace software. If you suspect that a sequencer capillary is blocked then inform the operator.

36 Noisy sequence due to overload signal : How to identify The sequencing trace shows noisy signal, preferably at the beginning of the trace. Raw data signals are saturated at approx units.

37 Noisy sequence due to overload signal : Traces A B Figure 1. Example of a trace with overload signal. (A) Processed data channel. (B) Raw channel data.

38 Noisy sequence due to overload signal : Causes Too much yield from the sequencing reaction and sequencing cleanup. Injection times are too long.

39 Noisy sequence due to overload signal : Troubleshooting Troubleshooting overloaded signals is easiest achieved by reducing the number of cycles in the cycle sequencing reaction or by adding additional EDTA in the eluted sequence. Injection times may be reduced in a re-run of the same samples.

40 n-1 primers: How to identify The sequence shows a pre-peak to every peak of the same colour of the following peak.

41 n-1 primers: Traces Figure 1: Tracefile showing n-1 peaks

42 n-1 primers: Causes n-1 primers are present in the sequencing primer, consisting of primers that are one base shorter than the original primer. n-1 primers are a common artifact of primer synthesis, but normally do not exceed 1% of all synthesized primer. If the number of n-1 primers represent more than 20-30% of the overall primers, this will result in measurable numbers of n-1 cycle sequencing products.

43 n-1 primers: Troubleshooting Change your oligo vendor or order only HPLC purified primers (recommended).

44 Mixed DNA templates : How to identify The sequencing trace has two or more sequence peaks at the same location. The secondary peaks may be the same height as the primary peaks down to about 20%. Lower levels of mixed signal (below 20%) are normally base called well by the KB. The trace signal strength is good (>200U) in the raw channel. The quality scores are low with often less than 100 Q20+ bases. The base called sequence does not match the expected sequence or any known sequence in GenBank.

45 Mixed DNA templates : Traces Figure 1. Example of a trace with mixed template. (A) Raw channel data. (B) Processed data channel at the same base location.

46 Mixed DNA templates : Causes Two or more templates were present in the reaction. This is the most common cause of mixed template. A "double pick" of two colonies. This can occur when the colonies are too close together on the colony plate. Two primers were present in the sequencing reaction. This can occur when using premixed PCR reagents for sequencing where the primer stock is actually a mix of universal forward and reverse oligonucleotide primers. The PCR fragment was not purified from PCR primes before sequencing. Two priming sites are present in DNA template. This can occur when a PCR product with universal priming tails is cloned into a plasmid or if there are sequence similarities in the templates. Poor quality PCR template containing multiple DNA fragments was used. Too low primer annealing temperature was used in the sequencing reaction.

47 Mixed DNA templates : Troubleshooting Makes sure only one DNA template is present. Prepare a new plasmid prep making sure that only one colony is selected. If sequencing a PCR product check that only one PCR product is present by running an agarose gel. Remember that even a relatively low amount of a small PCR product can cause mixed template problems and that gels cannot determine between fragents that are at least 10bp apart. Check the template for possible multiple priming sites. If two sites are present use a different primer. This can often occur when a fragment that contains the priming site is subcloned into a vector that also contains the priming site. Be aware of this problem when using the M13 universal primers. Insure that you use a PCR clean-up protocol that remove leftover PCR primers. Even low levels of the PCR primers can cause mixed signal problems, especially if they have a high annealing temperature. Check the predicted melting temperature of the sequencing primer. If it is more than 5 C above the annealing temperature used in the sequencing reaction then raise the annealing temperature. Because of the inclusion ditp in the BigDye sequencing mix the annealing/extension temperature can't be raised above 60 C. If your primer is still misanneals at 60 C then synthesize a new primer (this is easily done by removing bases from the 5' end until the Tm is below 60 C).

48 Short DNA Trace Read Lengths : How to identify Sequence provide less than 650 Q20+ bases for traces run on 36cm arrays and less than 750 Q20+ bases for traces collected on 50cm arrays under standard run conditions. End of the trace is "messy" with "rough" looking peaks Trace signal ends abruptly before base 800 on 36cm array traces and before base 1000 on 50cm array traces.

49 Short DNA Trace Read Lengths : Traces Figure 1. Example of a short/poor trace. (A) KB processed data channel data at approximately base 550. (B) Raw channel data at the same base location.

50 Short DNA Trace Read Lengths: Causes Too much or too little amount of template DNA Excessive dilution of the BigDye reagent Too much or too little amount of primer "Dirty" template DNA has been used Unsequencable region reached (eg. homopolymer G)

51 Short DNA Trace Read Lengths: Troubleshooting Check the concentration of the template DNA by gel electrophoresis. Do not rely on spectrophotometer readings alone as spec reading are often inaccurate. Check to see if an unsequencable region has been reached. If an unsequencable or hard stop region is the cause then it may be possible to sequence though it by PCR amplifying the region using 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP), then sequencing the PCR product directly. The 7-deaza-dGTP analog disrupts the hoogsteen base pairing between successive guanosine bases and allows the sequencing DNA polymerase to walk through the high G+C region. Check that the oligonucleotide primer concentration is correct. Do not rely on old primer stocks! Check if the template is clean. Consider sequencing a PCR amplified insert or switching to using a commercial plasmid miniprep kit if you are finding that many of your sequencing reactions are failing.

52 Traces with poorly resolved or "blurry" trace chromatogram peaks: How to identify The trace peaks are very broad or "blurry". This blurriness occurs before base 500 for trace collected using 36cm arrays, or before base 650 for traces collected on 50cm arrays. The nucleotide runs in the sequence appear as indistinct blobs rather than as individually separate peaks.

53 Traces with poorly resolved or "blurry" trace chromatogram peaks: Traces Figure 1. Example of a "blurry" or poorly resolved trace chromatogram collected using a ABI cm array.

54 Traces with poorly resolved or "blurry" trace chromatogram peaks: Causes Capillary overload. This is normally caused by running "dirty" samples with large amounts of template (or other) DNA, proteins or salt. High sequencing run voltages. The higher the run voltage the worse the resolution of the peaks in the trace file.

55 Traces with poorly resolved or "blurry" trace chromatogram peaks: Troubleshooting Use clean DNA. Avoid using templates that contain a significant amount of Genomic DNA, protein or salt. Check the concentration of the template. If the template concentration was too high then resequence using less template. Check that the correct run voltage was used. This can be determined by examining the run protocol contained with the ABI trace file.

56 DNA sequencing problems caused by excess free dye terminators: How to identify The trace has one or more large, broad peaks between bases 50 and 140. Each large peak color appears at different base positions as separate peaks.

57 DNA sequencing problems caused by excess free dye terminators: Traces Figure 1. Example of a trace with a moderate dye blob problem. Note that the base call is incorrect at approximately base 65. Figure 2. Example of a minor dye blob problem. Note that the base call is not compromised by this small dye peak.

58 DNA sequencing problems caused by excess free dye terminators: Causes Poor post-sequencing clean up. Does most frequently occur with EtOH precipitation and sephadex purifications. Not a problem with Agencourt CleanSEQ. Lost pellet. When the DNA pellet is lost during EtOH precipitation the remaining small amount of Big Dye can appear as large dye blob peaks. Failed reaction. When a reaction works inefficient or fails completely, the amount of leftover dye that was not incorporated during cycle sequencing can appear as large dye blob peaks. Note that with Agencourt CleanSEQ dye blobs commonly indicate that the sequencing reaction ran inefficiently!

59 DNA sequencing problems caused by excess free dye terminators: Troubleshooting Check the ethanol. 100% ethanol absorbs water from the air. Check to see if the sequencing reaction has failed by examining the raw trace data using software like FinchTV. If the reaction ran inefficient or failed then the dye blob problem is mostly likely only an artifact of having no or very little trace signal present.

60 DNA sequencing traces with noisy or weak signal : How to identify The trace peaks are misshapen and/or contain secondary peaks under the primary peak. The sequencing peaks are very weak (under 150 units) in the raw channel before base 300. The total quality scores counts are generally low.

61 DNA sequencing traces with noisy or weak signal : Traces Figure 1. Example of a noisy or weak trace. (A) Processed data channel. (B) Raw channel data. Note the misshapen secondary peaks in the processed channel and the very low signal strength (~100 units) in the raw channel.

62 DNA sequencing traces with noisy or weak signal : Causes Partially failed sequencing reaction. Too much or too little DNA. Any of the other causes listed in the failed DNA sequencing reaction chapter.

63 DNA sequencing traces with noisy or weak signal : Troubleshooting The solutions for solving weak signal traces are basically the same as those for solving failed reactions. For help on this problem see the solutions listed failed DNA sequencing reaction chapter.

64 DNA Sequencing Traces With Primer Dimers : How to identify The trace signal is mixed in the early regions (normally before base 400) yet the late regions are not mixed. The signal drops from very high early in the trace (usually in the first 100 bases) to much lower levels. Examination of the raw signal shows a high signal "block. The total quality scores counts are generally low to moderate depending on the region affected.

65 DNA Sequencing Traces With Primer Dimers : Traces Figure 1. DNA Sequencing Traces With Primer Dimers. Processed data channel. Note that the sequence is mixed up to base 80, then continues as a normal sequence.

66 DNA Sequencing Traces With Primer Dimers : Causes Contamination of the template, primer stock or other sequencing reagents with primer dimers. Too low annealing temperature. The sequencing primer is self-annealing. Sequencing TempliPhi templates. For reasons unknown TempliPhi templates are much more prone to primer dimer problems.

67 DNA Sequencing Traces With Primer Dimers : Troubleshooting Change all the sequencing reagents and water for a fresh batch. Also clean the pipettors as they can also be the cause of the primer dimer contamination. Raise the annealing temperature and/or use a shorter annealing time. For primer design, use a software that checks for self-annealing (like Primer 3).

68 Sharp peaks in the trace signal : How to identify The trace file contains sharp spikes (from 1 to 9 data points wide) in the raw data channel that can have intensities as high as 32,000 (detector limit). The sharp peaks are normally higher than the surrounding sequence peaks. The sharp peaks are generally more common towards the end of the trace than in the beginning but they can occur anywhere in the trace.

69 Sharp peaks in the trace signal : Traces Figure 1. Example of sharp signal spikes. (A) KB processed data channel data. (B) Raw channel data at the same location. (C) Large signal spike (raw channel). Note that the signal reaches an intensity over 32,000.

70 Sharp peaks in the trace signal : Causes The cause of sharp signal spikes is pretty much a mystery. The current best theory is that these peaks are caused by small gas bubbles forming in the capillaries during electrophoreses which refract the laser light into the CCD camera when they pass by the detector zone. The frequencies of sharp spikes seems to be operator and machine dependent. Troubleshooting The best solution is to purge buffers and load fresh matrix. Avoiding excessive shaking of the buffers may also help.

71 Multinucleotide repeat sequence slippage sequencing problems : How to identify The trace data becomes mixed after a long mononucleotide (single base) run in the DNA template. Can also occur on long dinucleotide repeats in a manner analogous to stutter bands that occur in microsatellite polymerase chain reaction (PCR) amplification.

72 Multinucleotide repeat sequence slippage sequencing problems : Traces Figure 1. Sequence slippage on a mononucleotide T run. Figure 2. Sequence slippage on a mononucleotide C run.

73 Multinucleotide repeat sequence slippage sequencing problems : Causes Long runs of a mono nucleotide base causes the DNA polymerase to "slip" on the template. This occurs by either the template or extension product looping out and rehybridizing and resulting in the generation of sequencing products of varying size. These sequencing products then appear as mixed signal in the trace downstream of the mono nucleotide run. Insertion and deletions in one allele. This can occur when two alleles are sequenced together (PCR templates) and one of the two mononucleotide runs has an InDel polymorphism.

74 Multinucleotide repeat sequence slippage sequencing problems : Troubleshooting Sequence the DNA template from both directions. For example, if the template shows slippage after using the forward primer then sequence the template from the reverse direction. You may need to make a custom primer to do this if the template is large. Use a custom primer designed to hybridize just outside the mononucleotide or dinucleotide run region. Use the sequence-by-mutagenesis (SAM) approach to avoid have long mononucleotide runs in your templates.

75 Sudden ending of DNA sequence signal (hard stops) : How to identify Good quality trace signal suddenly stops, or rapidly declines, in region of the trace that should be well resolved. The trace peak signal falls to under 150 units (on ABI 3730 instruments) in the raw channel after the stop region. The average quality scores after the "hard stop" region are generally low. Template has a high percentage of G and/or C nucleotides, especially in the region where the stop occured.

76 Sudden ending of DNA sequence signal (hard stops) : Traces Figure 1. Example of a sudden "hard stop" at a region of strong template secondary structure. (A) Processed data channel. (B) Raw channel data.

77 Sudden ending of DNA sequence signal (hard stops) : Causes Regions of strong secondary structure in the DNA template. These region can fold back on themselves and form hairpin structures that the sequencing polymerase can not pass through. Long runs of G or C nucleotides in the template. These can not only form hairpins with strong secondary structure, but can also form single stranded conformational structures that the the sequencing polymerase has problems passing through. Long template regions of guanidine (G runs) are particular problematic because of the substitution of ditp for dgtp in the BigDye sequencing mix. The BigDye sequencing DNA polymerase does not efficiently extend runs of multiple inosine residues causing the polymerase to stop in the G run regions.

78 Sudden ending of DNA sequence signal (hard stops) : Troubleshooting Use a 1:4 ratio mix of BigDye to dgtp Sequencing premix. This mix will help for high G+C templates. Add a small amount of dgtp nucleotide (~40µM) to the BigDye mix. This helps the sequencing polymerase pass through long G runs without causing major compression problems. PCR amplify the template using 7-deaza-GTP or ditp to reduce the strength of the secondary structure. This allows the sequencing polymerase to be able to pass through the region causing the hard stop. Use the sequence-by-mutagenesis (SAM) approach to avoid to have long mononucleotide runs in your templates.

79 DNA sequencing traces with indels : How to identify The trace trace is excellent before the indel region and mixed after the indel region. The trace often has leading or trailing 'shifted' peaks where a small peak is found before or after a large peak.

80 DNA sequencing traces with indels : Traces Figure 1. Example of indel trace. Note the mixed sequencing after the seven T run. Figure 2. Example of 'shifted' peaks due to a template indel. Note the small G peak preceding the large single G peak present after the nine T run.

81 DNA sequencing traces with indels : Causes Direct sequencing of PCR products derived from polymorphic templates. The most common cause of this is when directly sequencing PCR products amplified from diploid templates with heterozygous regions. Random mutation has occurred during the cloning process or during plasmid mini prep process (usually during the colony picking stage). This is much more likely to occur if the cloned template is toxic or unstable in the E. coli host.

82 DNA sequencing traces with indels : Troubleshooting Clone the PCR products before sequencing if the template is heterozygous. If the template is unstable in E. coli try cloning into a low copy vector or using a different strain. Sequence from the other direction using a reverse primer. This option is more viable for small inserts.

83 Traces containing chimeras and sequence rearrangements : How to identify The trace signal is high quality up to a point where it becomes mixed. Sometimes the mixed signals appear to be related to each other in a way that the peaks present are 'echoed' latter in the trace. The trace sequence data aligns to the expected data up to a point and then shows no further homology.

84 Traces containing chimeras and sequence rearrangements : Traces Figure 1. Example of a chimeric trace.

85 Traces containing chimeras and sequence rearrangements : Causes Cloning artifacts. Chimeras can result from cloning two or more unrelated DNA fragments at once in the same vector. 'Unstable' sequences like repeats or long mononucleotide runs. This is a common problem when cloning cdna sequences as the long poly A tails can be quite unstable in many vectors or E. coli strains. 'Toxic' sequences. This can occur if the gene cloned is expressed in E. coli and is toxic to the cell. This tends to occur more commonly in high copy number vectors and templates with low a G+C ratio. Strong secondary structure. Deletion can occur in regions of strong secondary structure. This is commonly seen when PCR products are cloned as the PCR process selects for these deletions artifacts.

86 Traces containing chimeras and sequence rearrangements : Troubleshooting Isolate and sequence another clone. This can solve the problem if the particular clone you have selected has undergone a rare mutation event. Sometime going back to the original plate and reelecting the same colony can solve the problem as the mutation may have occurred in liquid culture. Use a low copy number vector or grow the cells at 30 C. This can help maintain the stability of unstable or toxic sequences by reducing the selective pressure for mutation or rearrangement. Don't over-grow your cultures. The long the cells are grown for the higher the selection pressure for a deletion or rearrangement in the desired sequence. Try cloning using a different E. coli strain. This can sometime work especially if you original used a non-cloning strain such as BL21. Try adding some glucose to the growth media to inhibit expression of the LacZ promoter. This can help if the instability is being caused by leaky expression of the lacz operon.

87 Traces with delayed sequencing signal start : How to identify The first signal peak in the raw channel is found after scan 3200 for 50cm traces and scan 2700 for 36cm traces. Traces normally start between scan 1800 and 2200 for 50cm traces and scan 1500 and 1900 for 36cm traces. The signal in the processed channels is often of low resolution with wide peaks.

88 Traces with delayed sequencing signal start : Traces Figure 1. Example of a good (A) and a delayed start (B) 50cm trace. The raw channel is shown.

89 Traces with delayed sequencing signal start : Causes Capillary overload. Unlike a lot of DNA sequencing problems delayed peak signal has only one cause - capillary overload. The two most common causes of capillary overload are too much template DNA or dirty template DNA contaminated with proteins an/or salt. Troubleshooting Use less template DNA. Modern ABI capillary sequencers require far less template DNA than the old slab based sequencers. However, many researchers are still using protocols developed for the slab gel based system. Use cleaner DNA. Try changing or at least improving your template preparation protocol.

90 THX!