Lidstrom:Sequencing with GeneWiz

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Return to [[Lidstrom:Protocols|Protocols]]
 
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==Setting Up an Account==
 
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*Talk to the Lidstrom purchasing manager (currently Melissa).    [[User:Janet B. Matsen|Janet]] 2/22/12
 
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==Submitting Samples==
 
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*Using the correct template mass and primer concentrations is crucial.  If you submit less than the recommended ng of DNA, sequencing fails more often.
 
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*If you have a low concentration plasmid prep, you can increase the mass you send by sending 0.5 uL of 100 uM primers (the stock concentration) instead of 5 uL of 5 uM primers.
 
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*The form has a section for notes.  The notes you can enter are for you only, not the sequencing center.  If you want to communicate with the sequencing staff, put it in the comments box at the bottom.  [[User:Janet B. Matsen|Janet]] 2/22/12
 
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==Aligning/Understanding Results==
 
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*[[User:Janet B. Matsen|Janet]] recommends doing a multiple alignment in NCBI's Blast webpage.  Do not use the APE program's aligner, especially if you have a repetitive sequence.  In Blast, try varying stringency for the match criteria if you don't have perfect alignment or you don't align the whole sequencing read to your expected sequence. 
 
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**If you get an imperfect match or fail to match to your expected sequence, you can loosen the stringency requirements by selecting "somewhat dissimilar" or "more dissimilar" instead of "highly similar." 
 
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**Always have good labels in your FASTA files to make sure you don't mix things up.
 
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**If you are making variants of a construct, you can compare your sequencing results to all of the things you are making.  First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor.  Include descriptions for each e.g. "> SH3-FLS in pSB1A3".  Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box.  When the results are returned, click the "max score" button & it will put the match with the highest score first.  This is most likely your product, but a sequencing read can match to multiple designs depending on your project.
 
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==When Sequencing Fails==
==When Sequencing Fails==
*There are several ways it can fail & you can see the GeneWiz help for causes/solutions below.  You can also watch this [http://www.genewiz.com/file/dnaseq201/dnaseq201.html GeneWiz webinar].
*There are several ways it can fail & you can see the GeneWiz help for causes/solutions below.  You can also watch this [http://www.genewiz.com/file/dnaseq201/dnaseq201.html GeneWiz webinar].
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**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Non-Specific.pdf Non-specific]: multiple priming sites
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**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Non-Specific.pdf Non-specific]
***multiple are present on your DNA, or you accidentally have a mixture of DNA templates
***multiple are present on your DNA, or you accidentally have a mixture of DNA templates
***hairpin present
***hairpin present
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Poor%20Quality.pdf Poor quality]  
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Poor%20Quality.pdf Poor quality]  
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***high background: there is a clear sequence but there is also a background sequence. 
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****remnants of chromosomal DNA
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****primer degradation: recall that your sequencing is dependent on length.  So if you have one population of primers that is 20 bp and one that is 18 bp, they will give two separate signals.
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***top heavy: beginning sequence is strong; peaks may even be cut off.  As it gets longer, the signal dies off.  This is due to improper primer:template ratio; your primer may be "used up".
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/No%20Priming.pdf No priming]: lots of "N"s in sequencing result
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/No%20Priming.pdf No priming]: lots of "N"s in sequencing result
***too much, too little, or no DNA
***too much, too little, or no DNA
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***carry-over inhibitors in the reaction such as phenol, ethanol, EDTA, or salts
***carry-over inhibitors in the reaction such as phenol, ethanol, EDTA, or salts
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Early%20Termination.pdf Early termination]
**[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Early%20Termination.pdf Early termination]
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***hairpin present, repetitive sequence, or high GC content can terminate the rxn sequencing.  Note: they have several "difficult sequencing" protocols that can be used; select them in the form.
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**bubble in capillary
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***can appear as a smear.  You won't know when this happens, but customer support may be able to tell.
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*You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.
*You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.
*You can resequence as many as you like for 1/2 price and you have the choice to use new preps.  This allows you to use a fresh miniprep or primer batch.  If you want to submit fresh samples for 1/2 price, you may want to contact customer service and get approval.  Once I put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form and they told me that was ok, but I got mixed messages about this.  [[User:Janet B. Matsen|Janet 2/22/12]]
*You can resequence as many as you like for 1/2 price and you have the choice to use new preps.  This allows you to use a fresh miniprep or primer batch.  If you want to submit fresh samples for 1/2 price, you may want to contact customer service and get approval.  Once I put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form and they told me that was ok, but I got mixed messages about this.  [[User:Janet B. Matsen|Janet 2/22/12]]
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**the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.  
**the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.  
**you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.
**you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.
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== Miscellaneous ==
 
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*Will my sequences be online forever?
 
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**After three months, the data from your account will automatically be archived. If you would like the data for orders older than three months you may request a transfer of results back into your account.  All you would need to do is log into your account, go to "Order Status & Results", change the search conditions, select the orders you would like transferred by checking the box under the Transfer column and then hit the Transfer button. This will then automatically send our technical support department your request. You should be able to click on the order number an view the DNA names/primers; however, you won't have access to the trace files or sequences until you request that they are transferred back to your account.  -[[User:Janet B. Matsen|Janet]] 2/22/12 (from customer service chat)
 
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*Why don't they have VF2 and VR (the standard BioBrick primers) available at GeneWiz like they do for other common primers?
 
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**Janet (and others) have been lobbying for them to include these.  Stay tuned!  -[[User:Janet B. Matsen|Janet]] 2/22/12
 
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*How much does each reaction cost?
 
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**Our lab pays $6/rxn if sequencing 1-47 samples in an order OR $5/rxn if 48 or more samples are sequenced per order.  Some labs that sequence much more frequently (e.g. David Baker) pay $1.50/sequence less because of their volume.
 

Revision as of 10:25, 1 March 2012

When Sequencing Fails

  • There are several ways it can fail & you can see the GeneWiz help for causes/solutions below. You can also watch this GeneWiz webinar.
    • Non-specific
      • multiple are present on your DNA, or you accidentally have a mixture of DNA templates
      • hairpin present
    • Poor quality
      • high background: there is a clear sequence but there is also a background sequence.
        • remnants of chromosomal DNA
        • primer degradation: recall that your sequencing is dependent on length. So if you have one population of primers that is 20 bp and one that is 18 bp, they will give two separate signals.
      • top heavy: beginning sequence is strong; peaks may even be cut off. As it gets longer, the signal dies off. This is due to improper primer:template ratio; your primer may be "used up".
    • No priming: lots of "N"s in sequencing result
      • too much, too little, or no DNA
      • no primer or sub-optimal primer concentration
      • primer site not complementary
      • carry-over inhibitors in the reaction such as phenol, ethanol, EDTA, or salts
    • Early termination
      • hairpin present, repetitive sequence, or high GC content can terminate the rxn sequencing. Note: they have several "difficult sequencing" protocols that can be used; select them in the form.
    • bubble in capillary
      • can appear as a smear. You won't know when this happens, but customer support may be able to tell.
  • You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.
  • You can resequence as many as you like for 1/2 price and you have the choice to use new preps. This allows you to use a fresh miniprep or primer batch. If you want to submit fresh samples for 1/2 price, you may want to contact customer service and get approval. Once I put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form and they told me that was ok, but I got mixed messages about this. Janet 2/22/12
    • If you need to sequence another 1/2 price rxn, you should probably chat with customer service and they will put a note on the account. They usually tell you to put the name of who you chatted with on the form, too.
  • Whether or not your repeat will be successful depends on several variables such as:
    • the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.
    • you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.
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