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Submitting_to_SRA
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Submitting_to_SRA
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#This script was to make raw data from CDRF Dairy Fecal Microbiome ready for submission to NCBI
#Samples were demultiplex with primers removed and cleaned with kneaddata before submission
#Step 1 make project file
#---------------------------------------------------Project File----------------------------------------------------------------#
#contents of project.file
Username jvhagey
Last Hagey
First Jill
Email [email protected]
Center University of California, Davis
Type Institute
Ownership owner
website https://doi.org/10.5281/zenodo.4026848
ProjectName Differences between rumen sampling methods and the fecal microbiome.
ProjectTitle Rumen Sampling Methods Bias Microbial Communities Observed
Description Here we describe the differences between the fecal and rumen microbiome as well as differences in types of rumen sampling.
Grant id=XXX, agency= XX, title=XXXX
#Step 2 make mimarkpackage
#---------------------------------------------------Mimarkfile File----------------------------------------------------------------#
#!/bin/bash
##
#SBATCH --mem=6G
#SBATCH --time=1-18:0:0
#SBATCH -p production
#SBATCH -o /share/magalab/Jill/Depeters/Submit_SRA/Mothur_SRA.out
#SBATCH -e /share/magalab/Jill/Depeters/Submit_SRA/Mothur_SRA.err
module load mothur/1.39.5
cd /share/magalab/Jill/Depeters/CutAdapt_TrimmedBC/No_Prime/
mothur /share/magalab/Jill/Depeters/Submit_SRA/Make_SRA.bat
#Demulti_Fastq.file contains in part (comes from Demultiplexing):
282 282_Trim_R1.fastq.gz 282_Trim_R2.fastq.gz
283 283_Trim_R1.fastq.gz 283_Trim_R2.fastq.gz
284 284_Trim_R1.fastq.gz 284_Trim_R2.fastq.gz
285 285_Trim_R1.fastq.gz 285_Trim_R2.fastq.gz
#etc for all samples
#Step 3 add additional information into mimark file.
#---------------------------------------------------mimark File------------------------------------------------------------------------#
#Opened this file and then added the rest of the information based on metadata file
*sample_name *description *sample_title *seq_methods *organism *collection_date *env_biome *env_feature *env_material *geo_loc_name *host *lat_lon host_life_stage host_sex host_subject_id host_taxid samp_store_dur samp_store_temp samp_vol_we_dna_ext
282 Stomach_Tube Cow2549_Stomach_Tube_Day_9 F515 and R806 pooled PCR triplicate bovine gut metagenome 18-Nov-16 Dairy Cow Rumen Liquid_Solids USA:California:Davis """Bovine, Dairy Cow""" not collected Adult Female CowID_2549 9913 (Bos taurus) ~1 Month C
283 Stomach_Tube Cow2549_Stomach_Tube_Day_11 F515 and R806 pooled PCR triplicate bovine gut metagenome 18-Nov-16 Dairy Cow Rumen Liquid_Solids USA:California:Davis """Bovine, Dairy Cow""" not collected Adult Female CowID_2549 9914 (Bos taurus)
284 Stomach_Tube Cow2477_Stomach_Tube_Day_7 F515 and R806 pooled PCR triplicate bovine gut metagenome 18-Nov-16 Dairy Cow Rumen Liquid_Solids USA:California:Davis """Bovine, Dairy Cow""" not collected Adult Female CowID_2477 9915 (Bos taurus)
285 Stomach_Tube Cow2477_Stomach_Tube_Day_9 F515 and R806 pooled PCR triplicate bovine gut metagenome 18-Nov-16 Dairy Cow Rumen Liquid_Solids USA:California:Davis """Bovine, Dairy Cow""" not collected Adult Female CowID_2477 9916 (Bos taurus)
#etc for all samples
#Step 4 make SRA file for submission
#-----------------------------------------------------------SRA File-------------------------------------------------------------------#
#!/bin/bash
##
#SBATCH --mem=6G
#SBATCH --time=1-18:0:0
#SBATCH -p production
#SBATCH -o /share/magalab/Jill/Depeters/Submit_SRA/Mothur_SRA.out
#SBATCH -e /share/magalab/Jill/Depeters/Submit_SRA/Mothur_SRA.err
module load mothur/1.39.5
cd /share/magalab/Jill/Depeters/CutAdapt_TrimmedBC/No_Prime/
mothur /share/magalab/Jill/Depeters/Submit_SRA/Make_SRA.bat
#bat file
make.sra(file=/share/magalab/Jill/Depeters/Submit_SRA/Demulti_Fastq.file, project=/share/magalab/Jill/Depeters/Submit_SRA/project.file, mimark=/share/magalab/Jill/Depeters/Submit_SRA/Demulti_Fastq.tsv, platform=ILLUMINA, instrument=Illumina_MiSeq)
#Step 5 submit data from server to NCBI SRA
#---------------------------------------------------Submitting Files to SRA------------------------------------------------------------#
module load aspera-connect/3.5.1
#Write [email protected] to get the key file for upload
cd /share/magalab/Jill/Depeters/Submit_SRA/
ascp -i /share/magalab/Jill/Depeters/Submit_SRA/mothur1.private.openssh -QT -l100m -k1 -d /share/magalab/Jill/Depeters/CutAdapt_TrimmedBC/No_Prime/*.fastq.gz [email protected]:submit/Test/jvhagey
Where username is your NCBI username. If the upload to the test area succeeds you can proceed to upload to the Production area. The only change to your command line will be to change "Test" to "Production".
ascp -i /share/magalab/Jill/Depeters/Submit_SRA/mothur1.private.openssh -QT -l100m -k1 -d /share/magalab/Jill/Depeters/CutAdapt_TrimmedBC/No_Prime/*.fastq.gz [email protected]:submit/Production/jvhagey
# -d is a directory that contains fastq raw files and submission.xml files
ascp -i /share/tearlab/Maga/Jill/16s/Demultiplex_Before_QC/Mothur/<key> -QT -l100m -k1 -d /share/tearlab/Maga/Jill/16s/Demultiplex_Before_QC/Mothur/For_SRA_Upload/ [email protected]:submit/Production/jvhagey
#Do this last submit.ready is a blank file but it signals ncbi that submission of fastq files are complete
ascp -i /share/magalab/Jill/Depeters/Submit_SRA/mothur1.private.openssh -QT -l100m -k1 -d /share/magalab/Jill/Depeters/Submit_SRA/submit.ready [email protected]:submit/Production/jvhagey
#Sequences are avaliable at SRA accession study number SRP115649.