Tools of stLFR(Single Tube Long Fragment Reads) data analysis.
stLFR FAQs is directed to bgi-MGITech_Bioinfor@genomics.cn.
Download source code package from https://github.com/MGI-tech-bioinformatics/stLFR_V1.2.
July 14, 2020
1. keep the log and temp results in stLFR Docker.
Mar 31, 2020
1. add a Docker version of stLFR V1.2.
2. fix one BUG occur in SV detection.
Jan 10, 2020
1. Updated SV module (SV2.1, https://github.com/MGI-tech-bioinformatics/stLFR_SV2.1_module)
2. Novel summary report of HMTL format.
3. Redesigned pipeline structure and changed some parameters.
May 6, 2019
There are several updates in stLFR_v1.1 comparing with v1:
1. Users could use an alternative reference type (hg19 or hs37d5) in stLFR_v1.1 by --ref option instead of only hg19.
2. Updated CNV and SV detection tools are implied in stLFR_v1.1 for decreasing false discovery rate.
3. Three figures used for illustrating stLFR fragment distribution and coverage are added.
4. NA12878 benchmark VCF by GIAB is used for haplotype phasing error calculation.
Setup
1. Install docker follow the official website
https://www.docker.com/
2. Then do the following for the workflow:
docker pull rjunhua/stlfr_reseq_v1.2:v2
3. Download and unzip the database
From BGI Cloud Drive:
https://pan.genomics.cn/ucdisk/s/Jvmuii
Or from OneDrive:
https://dwz.cn/ZPlGA0eJ
Notes:
1. Please make sure that you run the docker container with at least 45GB memory and 30 CPU.
2. The input is sample list and output directory which descripted below (Main progarm arguments).
3. The log files and temp results are all left, so the memory must be double-checked.
Running
1. Please set the following variables on your machine:
(a) $DB_LOCAL: directory on your local machine that has the database files.
(b) $DATA_LOCAL: directory on your local machine that has the sequence data and "samplelist" file.
"samplelist" must follow the format descripted bellow,
and the *PATH* in "samplelist" must be absolute dicrtory of $DATA_LOCAL.
(c) $RESULT_LOCAL: directory for result.
2. Run the command:
docker run -d -P \
--name $STLFRNAME \
-v $DB_LOCAL:/stLFR/db \
-v $DATA_LOCAL:$DATA_LOCAL \
-v $RESULT_LOCAL:$RESULT_LOCAL \
rjunhua/stlfr_reseq_v1.2:v2 \
/bin/bash \
/stLFR/bin/stLFR_SGE \
$DATA_LOCAL/samplelist \
$RESULT_LOCAL
3. After report is generated:
docker rm $STLFRNAME
Download
----------------
1. download main scripts:
git clone git@github.com:MGI-tech-bioinformatics/stLFR_V1.2.git
2. download database and unzip into 'db':
For China mainland users, please using BGI Cloud Drive link:
https://pan.genomics.cn/ucdisk/s/3mmUzy
For other region users, please using OneDrive link:
https://dwz.cn/UKb9SRWU
3. download tools and unzip into 'tools':
For China mainland users, please using BGI Cloud Drive link:
https://pan.genomics.cn/ucdisk/s/ErYf6v
For other region users, please using OneDrive link:
https://dwz.cn/dVkXg7No
4. preinstall tools
More than 20 softwares/tools are used in this pipeline, and some of them are difficult to build static binary.
The tools directory descriped below is just an example. So, please make sure these softwares/tools are installed firstly.
(a). software/tool list
bam2depth, bwa, circos, convert(ImageMagick), gnuplot,
gatk4, java, picard, python2, python3, R, rtg-tools, samtools
(b). some packages are required for software/tool, such as:
R: ggplot2,
karyoploteR(https://bioconductor.org/packages/release/bioc/html/karyoploteR.html)
Python2: vcf, pysam, numpy
Python3: pysam
Running
----------------
1. Make sure 'SAMPLELIST' file is in a right format.
2. Run script with default parameters:
perl bin/stLFR_SGE -l SAMPLELIST -outputdir OUTPUTDIR
two demo stLFR libraries are provided for testing, and every library consists two lanes:
1. T0001-2:
http://ftp.cngb.org/pub/CNSA/data1/CNP0000387/CNS0057111/
2. T0001-4:
http://ftp.cngb.org/pub/CNSA/data1/CNP0000387/CNS0094773/
-list FILE
Name of input file. This is required.
Five columns in the list file, which the front three columns are required:
1. name : unique sample ID in this analysis
2. path : fastq path(s) for this sample split with colon(:)
3. barcode : sample-barcode for each path split with colon(:), 0 means all used
4. reffile : reference with index, two inner options are 'hg19' and 'hs37d5', NULL means 'hs37d5'
5. vcffile : dbsnp file, default is NULL
6. blacklist : black list file(BED format) for SV
7. controllist : sorted control list file(BEDPE format) for SV
eg: SAM1 /DATA/slide1/L01 1-4,5,7-9
SAM2 /DATA/slide1/L01:/DATA/slide2/L01 0:1-8 hg19
SAM3 /DATA/slide2/L02 0 REFERENCE/ref.fa DBSNP/dbsnp.vcf
SAM4 /DATA/slide2/L03 0 hs37d5 - BLACKLIST CONTROLLIST
After all analysis processes ending, you will get these files below:
1. HTML report: *_cn.html, *_en.html
2. raw data summary: *.fastqtable.xls
3. stLFR barcode summary: *.fragtable.xls
4. alignment summary: *.aligntable.xls
5. variant summary: *.varianttable.xls
6. haplotype phasing summary: *.haplotype.xls, *.haplotype.pdf
7. evaluation summary (NA12878): *.evaluation.xls
8. quality distrubution in cleanfq: *.Cleanfq.qual.png
9. base distribution in cleanfq: *.Cleanfq.base.png
10. depth distribution in alignment: *.Sequencing.depth.pdf
11. accumulated depth distribution: *.Sequencing.depth.accumulation.pdf
12. insert size distrubition: *.Insertsize.metrics.txt, *.Insertsize.pdf
13. GC bias distrubution: *.GCbias.metrics.txt, *.GCbias.pdf
14. Fragment coverage figure: *.frag_cov.pdf
15. Fragment length distribution figure: *.fraglen_distribution_min5000.pdf
16. Fragment per barcode distribution figure: *.frag_per_barcode.pdf
17. variant CIRCOS: *.circos.svg, *.circos.png, *.legend_circos.pdf
- If user has "Permission denied" problem in the process of running，you can use the command "chmod +x -R stLFR_v2.1/tools" to get executable permission of tools.
Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions：
The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.
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