Can You Upload Gz Files to Sra
How to use NCBI SRA Toolkit effectively?
- NCBI SRA toolkit is a fix of utilities to download, view and search large volume of high-throughput sequencing data from NCBI SRA database at faster speed
- SRA database has several accessions including, SRR (run accession for actual sequencing data for the item experiment), SRX (experiment accession representing the metadata for study, sample, library, and runs), SRP (study accession representing the metadata for sequencing study and projection abstract), SAMN/SRS (BioSample/SRA accession representing the metadata for biological sample).
Applications
- Effectively download the large book of loftier-throughput sequencing data (eg. FASTQ, SAM)
- Catechumen SRA file into other biological file format (eg. FASTA, ABI, SAM, QSEQ, SFF)
- Call up a small subset of large files (e.g. sequences, alignment)
- Search within SRA files and fetch specific sequences
To install the latest version of SRA toolkit, download the binaries/install scripts for your Os from here
# I am using Ubuntu Linux # download latest version of compiled binaries of NCBI SRA toolkit # (version 2.xi.3) for Ubuntu Linux # Compiled binaries for other Os visit: https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software $ wget https://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/2.11.three/sratoolkit.2.xi.three-ubuntu64.tar.gz # extract tar.gz file $ tar -zxvf sratoolkit.2.xi.three-ubuntu64.tar.gz # add binaries to path using export path or editing ~/.bashrc file $ consign PATH = $PATH:/dwelling house/ren/software/sratoolkit.two.11.three-ubuntu64/bin # Now SRA binaries added to path and prepare to employ Download SRA datasets using NCBI SRA toolkit
Notation: Current SRA toolkit version 2.ten.8 does non back up Aspera customer (ascp). Even though ascp tin can run with older versions, it will download the information past https fashion and not by FASP manner.
# download file: prefetch will download and relieve SRA file related to SRR accession in # the current directory under newly created SRA accession directory $ prefetch SRR5790106 # for a single file $ prefetch SRR5790106 SRR5790104 # multiple files # convert to FASTQ: fastq-dump will convert SRR5790106.sra to SRR5790106.fastq $ fastq-dump SRR5790106 # single file $ fastq-dump SRR5790106 SRR5790104 # multiple files # at present you tin also replace fastq-dump with fasterq-dump which is much faster # and efficient for big datasets # by default it will use six threads (-east option) $ fasterq-dump SRR5790106 # single file $ fasterq-dump SRR5790106 SRR5790104 # multiple files # for paired-finish data use --dissever-files (fastq-dump) and -Due south or --dissever-files (fasterq-dump) choice # employ --divide-3 option, if the paired-end reads are not properly arranged (e.m. some reads has lack of mate pair) $ fastq-dump --split-files SRR8296149 $ fasterq-dump -Due south SRR8296149 # download biological and technical reads (cell and sample barcodes) in case of single # prison cell RNA-seq (10x chromium) $ fasterq-dump -S --include-technical SRR12564282 # download alignment files (SAM) # brand sure corresponding accretion has alignment file at SRA database $ sam-dump --output-file SRR1236468.sam SRR1236468 Note With
fastq-dumpandfasterq-dump,prefetchpace is unncessary and you tin directly download sequence data in FASTQ format. Usingfastq-dumpstraight withoutprefetchwill be slow equally compared to first usingprefetchand and thenfastq-dump
parallel-fastq-dump
parallel-fastq-dump is a wrapper to fastq-dump, which makes fastq-dump to run parallel. In brief, information technology splits the file based on number of threads and run fastq-dump parallel. Read more than here
Install parallel-fastq-dump as conda install -c bioconda parallel-fastq-dump
# download paired-end RNA-seq data with 8 threads parallel - fastq - dump -- sra - id SRR17062757 -- threads 8 -- split - files -- gzip parallel-fastq-dump download FASTQ files (with gzip compression) faster as compared to fasterq-dump. When I compared the operation to download SRR17062757 (~25 Thousand paired-cease reads), parallel-fastq-dump took 2m36.257s and fasterq-dump took 3m13.182s (without gzip compression).
Batch download SRA datasets
- Sometimes, we demand to download hundreds or thousands of FASTQ files from the SRA database and information technology would exist inconvenient to straight utilize the SRA toolkit for batch download
- I have added a wrapper script for
fasterq-dumpinbioinfokit(v0.nine.seven or later) for easy download of a large number of FASTQ files from the SRA database - Check bioinfokit documentation for installation and documentation
- Download exam SRA accretion file containing accessions for both single and paired-stop FASTQ datasets
# tested on Linux and Mac. Information technology may not work on Windows >>> from bioinfokit.analys import fastq # batch download fastq files # make sure you have installed the latest version of NCBI SRA toolkit (version 2.ten.viii) and added binaries in the # organisation path >>> fastq . sra_bd ( file = 'sra_accessions.txt' ) # increase number of threads >>> fastq . sra_bd ( file = 'sra_accessions.txt' , t = 16 ) # utilize fasterq-dump customized options, you lot can see more options for fas terq-dump as # fasterq-dump -help fastq . sra_bd ( file = 'sra_accessions.txt' , t = 16 , other_opts = '--outdir temp --skip-technical' ) # multiple FASTQ (technical and biological) files from from # 10x chromium unmarried prison cell 3' RNA-seq information # if you provide file containing SRA accessions for 10x chromium # single cell 3' RNA-seq data, it will give multiple FASTQ files # for example, SRA accession SRR12564282 will give three FASTQ files # (sample barcode, cell barcode, and biological read FASTQ files) fastq . sra_bd ( file = 'path_to_sra_file' , t = 16 , other_opts = '--include-technical --split-files' ) Validation of downloaded SRA data integrity
It is essential to bank check the integrity and checksum of SRA datasets to ensure successful download
# download FASTQ file $ prefetch SRR5790104 # fastq-dump SRR5790104 # check integrity of downloaded SRR5790106.fastq file # output from vdb-validate should written report 'ok' and 'consistent' for all parameters # Notation: make certain you take .sra (not .cache) file for corresponding accession in # sra accession directory $ vdb-validate SRR5790104 2020-08-31T22:46:27 vdb-validate.two.10.8 info: Table 'SRR5790104.sra' metadata: md5 ok 2020-08-31T22:46:27 vdb-validate.2.x.8 info: Column 'ALTREAD': checksums ok 2020-08-31T22:46:29 vdb-validate.two.10.viii info: Column 'QUALITY': checksums ok 2020-08-31T22:46:xxx vdb-validate.2.10.8 info: Cavalcade 'READ': checksums ok 2020-08-31T22:46:30 vdb-validate.ii.10.8 info: Column 'X': checksums ok 2020-08-31T22:46:30 vdb-validate.2.x.eight info: Cavalcade 'Y': checksums ok 2020-08-31T22:46:30 vdb-validate.ii.10.8 info: Table 'SRR5790104.sra' is consistent Customized download of SRA datasets
Yous tin use SRA tools for customized output of large SRA datasets without downloading complete datasets (Notation: some options are not available in fasterq-dump)
# print start 10 reads from single-terminate FASTQ file # -Z choice volition print output on screen (STDOUT) $ fastq-dump -X 10 -Z SRR5790106 # save FASTQ file to specified directory $ fastq-dump -O temp SRR5790106 $ fasterq-dump -O temp SRR5790106 # compress FASTQ file gzip or bzip2 $ fastq-dump -O temp SRR5790106 $ fastq-dump --gzip SRR5790106 $ fastq-dump --bzip2 SRR5790106 # Annotation: --gzip or --bzip2 options are not available with fasterq-dump # Multithreading $ fasterq-dump -eastward 10 SRR5790106 Convert SRA data into other biological formats
SRA tools allow y'all to convert SRA files into FASTA, ABI, Illumina native (QSEQ), and SFF format
# convert to FASTA # you need to first download the FASTQ file to convert to FASTA file $ fastq-dump --fasta lx SRR5790106 # if you have paired-terminate FASTQ, utilise --dissever-files -fasta 60 # if you don't apply --separate-files for paired-ends, the reads will be merged from both ends # number 60 represents number of bases per line # Note: --fasta options is not available with fasterq-dump # catechumen to ABI (CSFASTA and QVAL) $ abi-dump SRR5790106 # catechumen to QSEQ # SRA database should have alignment information submitted for corresponding accession $ illumina-dump --qseq 2 SRR1236472 # 2 for paired-end and 1 for single-finish # catechumen to SFF # SFF is a binary file format related to 454 loftier-throughput sequencing $ sff-dump SRR996630 Search within SRA files
Yous can search specific sequences or subset of sequences in SRA files
# search within SRA files # output volition be sequence read IDs $ sra-search GATGCCGCGCC SRR5790104 Get read length from the SRA file,
# this assumes that read length is aforementioned for all reads as in unfiltered FASTQ files $ fastq-dump -X ane -Z SRR5790106 | sed -n '2p' | awk '{ impress length }' Read 1 spots for SRR5790106 Written i spots for SRR5790106 100 Annotation: For every SRA tools, y'all can bank check all options by providing -h parameter (eg. fasterq-dump -h)
- Differential gene expression analysis using DESeq2
References
- Understanding SRA Search Results
If yous have whatever questions, comments or recommendations, please email me at reneshbe@gmail.com
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