GOM Illumina – old Virtual Box analyses

Transferring info over from Google Docs. Here is some of the older work I was doing with the GOM Illumina data (note that these commands precede the most recent analysis, and I am not using any of this processed data for the final publication):

De-multiplexed individual samples in QIIME

QIIME 1.4 Virtual box on iMac. Seems like these commands allow no errors in the barcode, though.

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-1_1_trimmed.txt -o sl_out/KO_1_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-1_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_1.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-1_2_trimmed.txt -o sl_out/KO_1_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-1_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_1.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-2_1_trimmed.txt -o sl_out/KO_2_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-2_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_2.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-2_2_trimmed.txt -o sl_out/KO_2_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-2_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_2.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-3_1_trimmed.txt -o sl_out/KO_3_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-3_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_3.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-3_2_trimmed.txt -o sl_out/KO_3_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-3_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_3.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-4_1_trimmed.txt -o sl_out/KO_4_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-4_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_4.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-4_2_trimmed.txt -o sl_out/KO_4_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-4_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_4.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-5_1_trimmed.txt -o sl_out/KO_5_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-5_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_5.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-5_2_trimmed.txt -o sl_out/KO_5_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-5_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_5.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-6_1_trimmed.txt -o sl_out/KO_6_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-6_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_6.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-6_2_trimmed.txt -o sl_out/KO_6_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-6_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_6.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-7_1_trimmed.txt -o sl_out/KO_7_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-7_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_7.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-7_2_trimmed.txt -o sl_out/KO_7_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-7_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_7.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-8_1_trimmed.txt -o sl_out/KO_8_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-8_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_8.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-8_2_trimmed.txt -o sl_out/KO_8_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-8_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_8.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-9_1_trimmed.txt -o sl_out/KO_9_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-9_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_9.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-9_2_trimmed.txt -o sl_out/KO_9_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-9_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_9.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-10_1_trimmed.txt -o sl_out/KO_10_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-10_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_10.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-10_2_trimmed.txt -o sl_out/KO_10_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-10_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_10.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-11_1_trimmed.txt -o sl_out/KO_11_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-11_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_11.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-11_2_trimmed.txt -o sl_out/KO_11_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-11_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_11.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-12_1_trimmed.txt -o sl_out/KO_12_1/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-12_1_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_12.txt –barcode_type 5

split_libraries_fastq.py -i ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-12_2_trimmed.txt -o sl_out/KO_12_2/ -b ~/Desktop/Shared_Folder/fastx_trimmed_files/1926-KO-12_2_barcode.txt -m ~/Desktop/Shared_Folder/QIIME_mapping_files/1926_KO_12.txt –barcode_type 5

Then reran demultiplexing, this time using QIIME 1.5 Virtual box on iMac. Seems like these commands allow no errors in the barcode, though (I think these outputs are the ones listed in sl_out_nobarcodemismatch files)

Picking OTUS

Using UCLUST, 99% similarity with trie prefiltering on QIIME 1.5 VirtualBox on iMac

pick_otus.py -m uclust -s 0.99 -t -z -/home/qiime/Desktop/Shared_Folder/sl_out/GOM_illumina_demultiplexed_fwd_concat_1to12_1 -o /home/qiime/Desktop/Shared_Folder/GOM_uclust99_otus/

Next Steps

  1. Concat de-multiplexed samples (two separate combined files, PE-Fwd and PE-Rev)
    • Looks like there were a lot of barcodes in the reverse direction with one N character within the barcode; should probably re-run demultiplexing allowing for one mismatch (at least in reverse direction, anyway).
  2. Run OTU clustering – UCLUST at 99%
  3. Assign Taxonomy – PYNAST and SILVA reference database
  4. Diversity analyses in QIIME
    • Alpha Diversity – Rarefaction
    • Beta Diversity – PCoA, UPGMA clustering
    • OTU Network analyses, view in Cytoscape
    • New analyses released in QIIME 1.5.0
  5. Run OTUs through PhyloSift
    • 18S taxonomy assignments using SSU-align and tree-placement method (run on individual samples)
    • Edge PCOA and Squash Clustering in guppy, for multisample comparison
  6. Run Metatranscriptome data through PhyloSift
    • Does protein-coding taxonomy agree with rRNA data?
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