deletions | additions       diff --git a/16S rDNA Sequencing and Analysis (Organism Identification).md b/16S rDNA Sequencing and Analysis (Organism Identification).md  index 7188cc2..214dc87 100644  --- a/16S rDNA Sequencing and Analysis (Organism Identification).md  +++ b/16S rDNA Sequencing and Analysis (Organism Identification).md 
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After PCR is completed, confirm the PCR reaction worked by agarose gel electrophoresis, all controls behaved as expected, and that you have DNA fragments of the correct size (380bp).   ##Submit Samples for Sequencing  Very few single-researcher labs maintain Sanger sequencing capacity. However, there are a number of DNA sequencing facilities (commercial and academic)  that provide sequencing services for researchers. They will handle as few as 1 single sample, or will allow you to submit an unlimited number of samples, typically arrayed in a 96-well plate. You will typically provide both your PCR product as well as your PCR primers for sequencing. Each facility will have its own guidelines concerning DNA and primer concentration. Our lab uses the UC DNA Sequencing Facility-UC Davis http://dnaseq.ucdavis.edu. If a quick internet search does not reveal the presence of a Sequencing facility Facility  near you, most sequencing centers will allow you to ship samples to them for sequencing. ##Sanger Sequence Processing  Upon receiving Sanger reads from a sequencing facility, typically via email, it is necessary to do some pre-processing before they can be analyzed. These steps include  quality trim trimming  the reads, reverse complement complementing  the reverse sequence, align aligning  the reads and generate generating  a consensus sequence. There are very limited options for free software that allow the user to perform these steps. We recommend SeqTrace for the user who wants to see the trace and process the sequences manually. We have also created a script that will do all of these steps automatically, but does not allow you to adjust any of the parameters The choice of our script (easy, little control) versus SeqTrace (more complex, more control) will depend on the user and the project.  
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Confirm that you have Python version 2.x. You can do this by typing:  python --version  You should see something that looks like "Python 2.6.9" If you see Python 3.x, seek outside  help to invoke an earlier version directly. http://www.python.org/download/releases/ 
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3. The program is able to recognize forward and reverse reads from information in the file name if they are properly formatted.  + Go to Traces and click on Find and mark forward/reverse. The default setting looks for _F for forward and _R for reverse. This can be edited in the Project settings (you can pull it up by clicking on the picture of the tools at the top of the page) and changing the search strings under trace settings  + If the program is able to recognize the forward/reverse reads it will place an orange left pointing arrow in front of reverse reads and a blue right pointing arrow in front of forward reads. This step is not necessary to get a consensus sequence, it just makes organizing the reads easier.   4. Pull up the Project Settings by clicking on the picture of tools at the top of the page. Click on the Sequence Processing tab, under Sequence trimming unclick the Automatically trim sequence ends button. You should also decrease the Min. confidence score under Consensus settings. The default option is 30 which represents a 99.9% quality score, for many reads this will be too stringent and will not allow you to get enough overlap to create aquality  consensus sequence. A minimum confidence score between 15 and 25 is normally okay but tuning may be required depending on your read quality. 5. Group your forward and reverse reads by highlighting both of them and clicking Group selected forward/reverse files (under Traces)  6. Under Sequences go to Generate Finished Sequences and click on for all trace files. (you will need to redo this every time you change the project settings).  7. To view your consensus sequence, click on the read pair group and then click on the magnifying glass at the top of the page. You should see something like Figure X. 
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Use the drop down menus to set it to convert .abi files to .fastq. Upload a file and convert it. The converted file will save to your downloads folder under the name sample.fastq. If you are working with a lot of reads we recommend immediately renaming the files to match the original abi file name to avoid confusion.  ###Edit and Create a Consensus Sequence  Once all of your files are inthe  fastq format, move all of them to the Sanger_seq folder in which you saved the merge_sanger_16s.pl script. Use the terminal to navigate to within this folder. Then, type: cd Desktop/Sanger_seq  Then, to run the script, type: 
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  In the first case the merging happened however there may be some conflicting bases. The fewer the better. It can be an indication of how confident the user should be with the results. Since this is a very crude method it should be noted that there is no fancy algorithm behind the merge. There is a crude comparison for which we keep the base that had the highest quality score.  The In the  second outcome, the sequences were trimmed too much when doing the QC. The length of both sequences end to end was smaller than the fragment length that we are looking for. This is an indication of poor quality sequence and most users should not proceed (others can lower the quality threshold set by the script). The newly merged file will be saved as file1_merged.fasta and can be uploaded to BLAST for identification.