deletions | additions       diff --git a/16S rDNA Sequencing and Analysis (Organism Identification).md b/16S rDNA Sequencing and Analysis (Organism Identification).md  index 2c0a759..4e49650 100644  --- a/16S rDNA Sequencing and Analysis (Organism Identification).md  +++ b/16S rDNA Sequencing and Analysis (Organism Identification).md 
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##Direct PCR (if not extracting DNA)  Centrifuge 1 ml of the overnight culture until the cells form a pellet at the bottom of the tube (about 5 minutes at 10,000 g), pour off the liquid on top (a.k.a. the supernatant) and resuspend the pellet  in 100 ul of sterile DNAase-free water. Incubate the samples at 100 deg C for 10 minutes to help lyse the cells. Use the resulting solution as the template in the PCR reaction below. ##PCR reaction  This reaction uses the 27F (AGAGTTTGATCMTGGCTCAG) and 1391R (GACGGGCGGTGTGTRCA) primers which amplify a near full-length bacterial (and many archaeal) 16S rRNA gene. Our lab uses standard PCR reagents (Qiagen or Kappa), with an annealing temperature of 54 deg C and an extension at 72 deg C of 90 seconds. Do not forget to include positive (any sample containing bacterial genomic DNA) and negative (e.g., just water) controls.  
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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 (~1350bp).   ##Submit Samples for Sequencing  Very few single-researcher labs maintain currently have the capacity to do  Sanger sequencing capacity. sequencing.  However, there are a number of DNA sequencing facilities (commercial and academic) that provide Sanger  sequencing services for researchers. They will handle as little as a single sample, or will allow you to submit an unlimited number of samples, typically arrayed in 96-well plates. You will typically provide both your PCR product as well asyour PCR  primers for sequencing. Don't sequencing (sometimes the same primers as used for PCR are used for sequencing). To get the most data, do not  forget to submit rquest  forward (27F) (e.g., using primer 27F)  and reverse (1391R) (e.g., using primer 1391R)  reactions for each sample. Each facility will have its own guidelines concerning DNA and primer concentration. Our lab uses the UC Davis DNA Sequencing Facility http://dnaseq.ucdavis.edu. If a quick internet search does not reveal the presence of a Sequencing Facility near you, most sequencing centers will allow you to ship samples to them for sequencing. ##Sanger Sequence Processing  The end product of Sanger sequencing is the production of sequence "reads" for each sample submitted.  Upon receiving Sanger reads from a sequencing facility, typically via e-mail, it is necessary to do some pre-processing before they can be analyzed. These steps include quality trimming the reads, reverse complementing the reverse sequence, aligning the reads, generating a consensus sequence, and converting to FASTA format. There are very limited options for free software that allow the user to perform these steps. In this workflow we recommend using an automated pipeline available at the Ribosomal Database Project \cite{Cole_2013} if working with a large number of sequences. This pipeline only provides a rough view, since it doesn't complement or align the reads, it simply quality trims them and outputs the data in a format that can be fed directly to the BLAST program at NCBI \cite{2231712}. This will at least give an idea of what genera, and sometimes which species, each sample belongs to. We then recommend processing samples of interest using SeqTrace \cite{stucky2012seqtrace} which allows the user to see the trace, process the sequences manually, and a get a longer, more accurate sequence for analysis.