This is the repository for the manuscript "Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system" written by Patrick D. Schloss, Sarah L. Westcott, Matthew L. Jenior, and Sarah K. Highlander.The raw data can be obtained from the Sequence Read Archive at NCBI under accession SRP051686, which are associated with BioProject PRJNA271568 and the processed movies can be obtained using the commands in mothur_raw.bash. The entire manuscript can be generated on a Mac OS X or Linux computer by running the following commands:
git clone https://github.com/SchlossLab/PacBio_16S.git
cd PacBio_16S
sh write.paper
In running these commands we assume that you have a copy of R and mothur installed in your PATH. You can then convert the resulting markdown file to a Word docx file by doing:
sh compile_docx.sh PacBioManuscript.md
open PacBioManuscript.docx
We ran 8 SMRT cells and their data were located in the following folders that
were housed within a folder called raw_data:
B01_1_Cell2_PacBioRun109_PSchloss311_p15
C01_1_Cell3_PacBioRun109_PSchloss315_p16
D01_1_Cell4_PacBioRun108_PSchloss310_p13
D01_1_Cell4_PacBioRun109_PSchloss316_p4
E01_1_Cell5_PacBioRun108_PSchloss311_p15
E01_1_Cell5_PacBioRun109_PSchloss317_p19
F01_1_Cell6_PacBioRun108_PSchloss312_p35
H01_1_Cell8_PacBioRun112_PSchloss319_p19
If you substitute the "_p" for "_v" you'll see what region each run represented. There were two V15 runs and two V19 runs. These regions were amplified with the following primer sets:
V19 AGRGTTTGATYMTGGCTCAG GGYTACCTTGTTACGACTT
V16 AGRGTTTGATYMTGGCTCAG ACRACACGAGCTGACGAC
V15 AGRGTTTGATYMTGGCTCAG CCCGTCAATTCMTTTRAGT
V13 AGRGTTTGATYMTGGCTCAG ATTACCGCGGCTGCTGG
V35 CCTACGGGAGGCAGCAG CCCGTCAATTCMTTTRAGT
V4 GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT
We also put sequencing indices at the 5' and 3' of the inserts to correspond to the different samples:
mock1 ccaac ccaac
mock2 ggttg ccaac
mock3 ttggt ccaac
mouse1 aggtg ccaac
mouse2 cttac ccaac
mouse3 gaact ccaac
human1 tccga ccaac
human2 acagt ccaac
human3 cactg ccaac
soil1 aacca ccaac
soil2 tgtca ccaac
soil3 aaacc ccaac
Putting it all together in an oligos file (pacbio.oligos), we get...
primer AGRGTTTGATYMTGGCTCAG GGYTACCTTGTTACGACTT v19
primer AGRGTTTGATYMTGGCTCAG ACRACACGAGCTGACGAC v16
primer AGRGTTTGATYMTGGCTCAG CCCGTCAATTCMTTTRAGT v15
primer AGRGTTTGATYMTGGCTCAG ATTACCGCGGCTGCTGG v13
primer CCTACGGGAGGCAGCAG CCCGTCAATTCMTTTRAGT v35
primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT v4
barcode ccaac ccaac mock1
barcode ggttg ccaac mock2
barcode ttggt ccaac mock3
barcode aggtg ccaac mouse1
barcode cttac ccaac mouse2
barcode gaact ccaac mouse3
barcode tccga ccaac human1
barcode acagt ccaac human2
barcode cactg ccaac human3
barcode aacca ccaac soil1
barcode tgtca ccaac soil2
barcode aaacc ccaac soil3