Scripts and other things for RNA-Seq

0_installSoftware.sh

# Add yourself to the sudo group
su -
usermod -aG sudo danny
exit

# Install the virtual box guest additions
cd /media/cdrom0
sudo sh ./VBoxLinuxAdditions.run

# Install R and deps
sudo apt install r-base
sudo nano /etc/apt/sources.list
sudo apt install libssl-dev
sudo apt install libxml2-dev
sudo apt install libcurl4-openssl-dev

# Install R packages
sudo R
if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("GenomicFeatures")
BiocManager::install("preprocessCore")
q("no")

# Install Trimmomatic
sudo apt install git
sudo apt install ant

mkdir software
cd software

git clone https://github.com/usadellab/Trimmomatic.git
cd Trimmomatic
ant

# Install STAR
git clone https://github.com/alexdobin/STAR.git
cd STAR/source
# [UPDATE 2024] - Checkout an older version of STAR
git checkout ee50484
make

# Install picard
git clone https://github.com/broadinstitute/picard.git
cd picard
# [UPDATED 2024] - Checkout an older version of PICARD
git checkout 5db8017
./gradlew shadowJar

# Install HTSlib / samtools / bcftools
sudo apt install autoconf
git clone https://github.com/samtools/htslib.git
git clone https://github.com/samtools/samtools.git
git clone https://github.com/samtools/bcftools.git

# Install HTSlib
cd htslib
git submodule update --init --recursive
autoreconf -i
./configure
make

# Install samtools
cd samtools
autoheader
autoconf -Wno-syntax
./configure
make

# Install bcftools
cd bcftools
autoheader
autoconf -Wno-syntax
./configure
make

# GATK install
wget https://github.com/broadinstitute/gatk/releases/download/4.2.6.1/gatk-4.2.6.1.zip
unzip gatk-4.2.6.1.zip

# SRA
wget https://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/3.0.0/sratoolkit.3.0.0-centos_linux64-cloud.tar.gz
mkdir sratoolkit
tar -xzf sratoolkit.3.0.0-centos_linux64-cloud.tar.gz –C sratoolkit
./sratoolkit/usr/local/ncbi/sra-tools/bin/vdb-config --interactive

# Make symbolic links
mkdir bin
cd bin
ln -s /home/danny/software/STAR/source/STAR STAR
ln -s /home/danny/software/htslib/bgzip bgzip
ln -s /home/danny/software/samtools/samtools samtools
ln -s /home/danny/software/bcftools/bcftools bcftools
ln -s /home/danny/software/sratoolkit/usr/local/ncbi/sra-tools/bin/fasterq-dump fasterq-dump

#Update the bashrc file
nano ~/.bashrc

# Add at the end:
export PATH="$HOME/bin:$PATH"

# Additional link:
ln -s /home/danny/software/htslib/tabix tabix
# Additional R package:
sudo R
install.packages("ggplot2")
install.packages("gplots")
install.packages("gsalib")
q("no")

wget https://data.broadinstitute.org/igv/projects/downloads/2.14/IGV_Linux_2.14.1_WithJava.zip
unzip IGV_Linux_2.14.1_WithJava.zip

1_buildGenome.R

#
# Download Saccharomyces Cerevisiae genome
# copyright (c) 2022 - Danny Arends
#

uri <- "ftp.ensembl.org/pub/release-108/fasta/saccharomyces_cerevisiae/dna/"
base <- "Saccharomyces_cerevisiae.R64-1-1.dna.chromosome."
chrs <- c(as.character(as.roman(seq(1:16))), "Mito")

# Download
for(chr in chrs){
  fname <- paste0(base, chr, ".fa.gz")

  # Download command
  cmd <- paste0("wget ", uri, fname)
  #cat(cmd, "\n")
  system(cmd)
}


# Create an empty the file
cat("", file = "Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa")
for(chr in chrs){
  fname <- paste0(base, chr, ".fa.gz")
  # Extract and merge into a fast file
  cmd <- paste0("zcat ", fname, " >> Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa")
  #cat(cmd, "\n")
  system(cmd)
}

# Compress the fasta file using bgzip (keep original)
cmd <- paste0("bgzip -k Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa")
#cat(cmd, "\n")
system(cmd)

# Delete the chromosomes
for(chr in chrs){
  fname <- paste0(base, chr, ".fa.gz")
  # Extract and merge into a fast file
  cmd <- paste0("rm ", fname)
  #cat(cmd, "\n")
  system(cmd)
}

2_prepGenome.sh

# Get the reference transcriptome and GTF for STAR
wget http://ftp.ensembl.org/pub/release-108/gtf/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.108.gtf.gz
gunzip -k Saccharomyces_cerevisiae.R64-1-1.108.gtf.gz
wget http://ftp.ensembl.org/pub/release-108/variation/vcf/saccharomyces_cerevisiae/saccharomyces_cerevisiae.vcf.gz

# Index the genome using samtools
samtools faidx Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa.gz

# Generate genome/transcriptome index using STAR
STAR --runThreadN 2 --runMode genomeGenerate \
     --genomeDir ~/genome/STAR \
     --genomeSAindexNbases 10 \
     --sjdbGTFfile ~/genome/Saccharomyces_cerevisiae.R64-1-1.108.gtf \
     --genomeFastaFiles ~/genome/Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa

# Get the reference SNPs and index using tabix
tabix saccharomyces_cerevisiae.vcf.gz

# Index the genome using picard
java -Xmx4g -jar /home/danny/software/picard/build/libs/picard.jar CreateSequenceDictionary \
     -R Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa.gz

3_pipeline.R

#
# Align SRA reads to the Saccharomyces Cerevisiae genome
# copyright (c) 2022 - Danny Arends
#

execute <- function(x, outputfile = NA, intern = FALSE, quitOnError = FALSE){
  if(!is.na(outputfile) && file.exists(outputfile)){
    cat("Output for step exists, skipping this step\n");
    invisible("")
  }
  cat("----", x, "\n"); res <- system(x, intern = intern); cat(">>>>", res[1], "\n")
  if(res[1] >= 1){ 
    cat("Error external process did not finish\n\n");
    if(quitOnError) q("no")
  }
}

input.dir <- "/home/danny/data/raw"
input.base <- "SRR13978643"  #Get from the command line
output.dir <- paste0("/home/danny/data/output/", input.base,".aln")
genome.path <- "/home/danny/genome/STAR"
ref.fa.gz <- "/home/danny/genome/Saccharomyces_cerevisiae.R64-1-1.dna.primary_assembly.fa.gz"
ref.snps <- "/home/danny/genome/saccharomyces_cerevisiae.vcf.gz"

# Create an output folder
if(!file.exists(input.dir)){ dir.create(input.dir, recursive = TRUE) }
if(!file.exists(output.dir)){ dir.create(output.dir, recursive = TRUE) }

# STEP 0 - SRA Download and Compress
setwd(input.dir)

execute(paste0("fasterq-dump -p --split-files ", input.base), paste0(input.base, "_1.fastq"))
execute(paste0("bgzip ", input.base, "_1.fastq"), paste0(input.base, "_1.fastq.gz"))
execute(paste0("bgzip ", input.base, "_2.fastq"), paste0(input.base, "_2.fastq.gz"))

# STEP 1 - READ Trimming
trim.files  <- c(
                  paste0(input.dir, "/", input.base,"_1.fastq.gz"),
                  paste0(input.dir, "/", input.base,"_2.fastq.gz"),
                  paste0(output.dir, "/", input.base,"_1.P.fastq.gz"),
                  paste0(output.dir, "/", input.base,"_1.U.fastq.gz"),
                  paste0(output.dir, "/", input.base,"_2.P.fastq.gz"),
                  paste0(output.dir, "/", input.base,"_2.U.fastq.gz")
                )
trim.path <- "/home/danny/software/Trimmomatic"
trim.exec <- paste0("java -jar ", trim.path, "/dist/jar/trimmomatic-0.40-rc1.jar")
trim.opts <- paste0("ILLUMINACLIP:",trim.path,"/adapters/TruSeq3-PE-2.fa:2:30:10")
trim.opts <- paste0(trim.opts, " LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36")
trim.cmd  <- paste0(trim.exec, " PE ", paste0(trim.files, collapse=" "), " ", trim.opts)

execute(trim.cmd, trim.files[3])

# STEP 1.1 - UNZIP for STAR
execute(paste0("gunzip -k ", trim.files[3]), gsub(".fastq.gz", ".fastq", trim.files[3]))
execute(paste0("gunzip -k ", trim.files[5]), gsub(".fastq.gz", ".fastq", trim.files[5]))

files.in <- gsub(".fastq.gz", ".fastq", trim.files[c(3,5)])

# STEP 2 - Alignment using STAR
star.outbase <- paste0(output.dir, "/", input.base)
star.bam <- paste0(star.outbase, "Aligned.sortedByCoord.out.bam")

star.exec <- "STAR --runMode alignReads"
star.opts <- paste0("--genomeDir ", genome.path, " --outSAMtype BAM SortedByCoordinate")
star.in <- paste0("--readFilesIn ", paste0(files.in, collapse=" "))
star.out <- paste0("--outFileNamePrefix ", star.outbase)
star.cmd  <- paste0(star.exec, " ", star.in, " ", star.opts, " ", star.out)

execute(star.cmd, star.bam)

# STEP 2.1 - Create a samtools index
execute(paste0("samtools index ", star.bam), paste0(star.bam, ".bai"))
# STEP 2.2 - Create mapping and coverage statistics
execute(paste0("samtools flagstats ", star.bam))
execute(paste0("samtools coverage ", star.bam))

#STEP 3 - Remove duplicate reads using picard tools
p.bam <- paste0(star.outbase, "Aligned.sortedByCoord.RD.out.bam")
metrics.out <- paste0(star.outbase, "_metrics.txt")

p.exec <- "java -Xmx4g -jar /home/danny/software/picard/build/libs/picard.jar"
p.in <- paste0("-I ", star.bam)
p.out <- paste0("-O ", p.bam, " -M ", metrics.out)
p.opts <- paste0("--REMOVE_DUPLICATES true")
p.cmd <- paste0(p.exec, " MarkDuplicates ", p.opts," ", p.in, " ", p.out)

execute(p.cmd, p.bam)

# STEP 3.1 - Create a samtools index
execute(paste0("samtools index ", p.bam), paste0(p.bam, ".bai"))
# STEP 3.2 - Create mapping and coverage statistics
execute(paste0("samtools flagstats ", p.bam))
execute(paste0("samtools coverage ", p.bam))

# STEP 4 - Add read group (1) and sample run, library, and name
rg.bam <- paste0(star.outbase, "Aligned.sortedByCoord.RD.RG.out.bam")
rg.opts <- paste0("-PL ILLUMINA -PU run -LB ", gsub("SRR", "", input.base), " -SM ", input.base)
p.cmd <- paste0(p.exec, " AddOrReplaceReadGroups -I ", p.bam, " -O ", rg.bam, " ", rg.opts)
execute(p.cmd)

# STEP 4.1 - Create a samtools index
execute(paste0("samtools index ", rg.bam), paste0(rg.bam, ".bai"))

# STEP 5 - GATK prep
gatk.exec <- "java -Xmx4g -jar /home/danny/software/gatk-4.2.6.1/gatk-package-4.2.6.1-local.jar"
gatk.opts <- paste0("-R ", ref.fa.gz, " --known-sites ", ref.snps)

# STEP 5.1 - GATK BaseRecalibrator
gatk.cov1 <- paste0(star.outbase, "_cov1.txt")
gatk.cmd  <- paste0(gatk.exec, " BaseRecalibrator ", gatk.opts, " -I ", rg.bam, " -O ", gatk.cov1)
execute(gatk.cmd, gatk.cov1)

# STEP 5.2 - GATK ApplyBQSR
recal.bam <- paste0(star.outbase, "Aligned.sortedByCoord.RD.RG.RC.out.bam")
gatk.cmd  <- paste0(gatk.exec, " ApplyBQSR -R ", ref.fa.gz, " -bqsr ", gatk.cov1, " -I ", rg.bam, " -O ", recal.bam)
execute(gatk.cmd, recal.bam)

# STEP 5.3 - GATK BaseRecalibrator
gatk.cov2 <- paste0(star.outbase, "_cov2.txt")
gatk.cmd  <- paste0(gatk.exec, " BaseRecalibrator ", gatk.opts, " -I ", recal.bam, " -O ", gatk.cov2)
execute(gatk.cmd, gatk.cov2)

# STEP 5.4 - GATK AnalyzeCovariates
recal.plot <- paste0(star.outbase, "AnalyzeCovariates.pdf")
gatk.cmd  <- paste0(gatk.exec, " AnalyzeCovariates -before ", gatk.cov1, " -after ", gatk.cov2, "  -plots ", recal.plot)
execute(gatk.cmd)

# STEP 6 - Index the recalibrated bam files
execute(paste0("samtools index ", recal.bam), paste0(recal.bam, ".bai"))

# STEP 6.1 - Create mapping and coverage statistics
execute(paste0("samtools flagstats ", recal.bam))
execute(paste0("samtools coverage ", recal.bam))

q("no")

Hi Danny,
In the buildGenome.sh, the uri uses the release-107, whereas in the prepGenome.sh, we used the release-108, was this intentional?

Hi Danny, In the buildGenome.sh, the uri uses the release-107, whereas in the prepGenome.sh, we used the release-108, was this intentional?

@sunilnahata No, both should be 108, the issue is the 108 version was released during last week when I was still working on the code, I'll update it to make sure both are the same

Hi Danny, In the buildGenome.sh, the uri uses the release-107, whereas in the prepGenome.sh, we used the release-108, was this intentional?

No, both should be 108, the issue is the 108 version was released during last week when I was still working on the code, I'll update it to make sure both are the same

Oh, alright. Thanks again for your session today, it was quite a learning experience.

Hi Danny,
I think there is a misplaced = sign in the "--genomeDir=" option in the following line
star.opts <- paste0("--genomeDir=", genome.path, " --outSAMtype BAM SortedByCoordinate")
It was throwing me an error. I removed it and put a space instead and it worked.

Hi Danny, I think there is a misplaced = sign in the "--genomeDir=" option in the following line star.opts <- paste0("--genomeDir=", genome.path, " --outSAMtype BAM SortedByCoordinate") It was throwing me an error. I removed it and put a space instead and it worked.

This is indeed weird, IIRC it shouldn't matter if there is an = separating the command-line option name and the value, but t's better for consistency to have them all without. Looking through my old code I see many instances of using an = and I've never encountered an issue with it. according to the getopt specs, they should be equivalent (https://en.wikipedia.org/wiki/Getopt).

Anyway, fixed it to be consistent

Hi Danny, I think there is a misplaced = sign in the "--genomeDir=" option in the following line star.opts <- paste0("--genomeDir=", genome.path, " --outSAMtype BAM SortedByCoordinate") It was throwing me an error. I removed it and put a space instead and it worked.

This is indeed weird, IIRC it shouldn't matter if there is an = separating the command-line option name and the value, but t's better for consistency to have them all without. Looking through my old code I see many instances of using an = and I've never encountered an issue with it. according to the getopt specs, they should be equivalent (https://en.wikipedia.org/wiki/Getopt).

Anyway, fixed it to be consistent

Yeah. It seems like Picard also changed its syntax. They now require = sign instead of the space for specifying the options. Ideally it shouldn't matter. I don't know why they're doing this. Anyways, thanks a lot for these tutorials. They're really helpful.

Hi Danny! If my raw files are on my computer, how can i input it into debian and how should i modify the execute code? Thank you so much!!

Hi Danny! If my raw files are on my computer, how can i input it into debian and how should i modify the execute code? Thank you so much!!

You can mount a shared folder in Virtualbox and copy / read the files from there. First install the "VirtualBox Guest Additions tool" (We did this in line 7 - 8), then close the virtual box and add a shared folder:

• Select / Navigate to Machine > Settings.
• In the “Settings” window, switch to the “Shared Folders” tab.
• Click the “Add” button to create a new shared folder.

Hi Danny! If my raw files are on my computer, how can i input it into debian and how should i modify the execute code? Thank you so much!!

You can mount a shared folder in Virtualbox and copy / read the files from there. First install the "VirtualBox Guest Additions tool" (We did this in line 7 - 8), then close the virtual box and add a shared folder:

• Select / Navigate to Machine > Settings.
• In the “Settings” window, switch to the “Shared Folders” tab.
• Click the “Add” button to create a new shared folder.

I tried that in debian but it showed me (sh: 0: cannot open ./VBoxLinuxAdditions.run: No such file)

If the file is not found it, means that you didn't mount the VirtualBox additions CD. See the process explained at 35 minutes into the video. In short: download the additions CD, mount the CD, then execute lines 1 through 8

After that shutdown the machine and add a shared folder, as described in my previous comment.

If the file is not found it, means that you didn't mount the VirtualBox additions CD. See the process explained at 35 minutes into the video. In short: download the additions CD, mount the CD, then execute lines 1 through 8

After that shutdown the machine and add a shared folder, as described in my previous comment.

Thanks! i will take a look! Really appreciate your help!

Dear Danny, Thank you for the video.

I am trying to run step 3 (Remove duplicate reads using picard tools) of this lecture, but it's showing an error message at 1:30:00 of the video. I have tried to look for a solution online, but none is providing a solution to the problem. I would appreciate your help. Thank you.

[Sun Jan 14 14:55:18 CST 2024] Executing as rufus@debian on Linux 6.1.0-17-amd64 amd64; OpenJDK 64-Bit Server VM 17.0.9+9-Debian-1deb12u1; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: Version:3.1.1-8-ga638ce92a-SNAPSHOT
INFO 2024-01-14 14:55:18 MarkDuplicates Start of doWork freeMemory: 38556984; totalMemory: 50331648; maxMemory: 4294967296
INFO 2024-01-14 14:55:18 MarkDuplicates Reading input file and constructing read end information.
INFO 2024-01-14 14:55:18 MarkDuplicates Will retain up to 15561475 data points before spilling to disk.
WARNING 2024-01-14 14:55:19 AbstractOpticalDuplicateFinderCommandLineProgram A field field parsed out of a read name was expected to contain an integer and did not. Read name: SRR13978643.342872. Cause: String 'SRR13978643.342872' did not start with a parsable number.
[Sun Jan 14 14:55:19 CST 2024] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 0.02 minutes.
Runtime.totalMemory()=241172480
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" java.lang.NullPointerException: Cannot invoke "htsjdk.samtools.SAMReadGroupRecord.getReadGroupId()" because the return value of "htsjdk.samtools.SAMRecord.getReadGroup()" is null
at picard.sam.markduplicates.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:558)
at picard.sam.markduplicates.MarkDuplicates.doWork(MarkDuplicates.java:270)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:280)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:105)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:115)

1
Error external process did not finish

No idea at first sight, but it seems to have to do with the bam file your supplying not having a numerical value for its ReadGroup. See if the ReadGroup is a numeric value by using samtool. https://gatk.broadinstitute.org/hc/en-us/articles/360035890671-Read-groups Does the BAM file work in other programs, such as the IGV viewer? If not, try to regenerate the BAM by running the previous step and looking for warnings. Good luck, debugging Sent from Outlook for Android<https://aka.ms/AAb9ysg>

…

________________________________ From: Olamide Adesina ***@***.***> Sent: Sunday, January 14, 2024 9:52:22 PM To: Olamzq ***@***.***> Cc: Author ***@***.***>; Comment ***@***.***> Subject: Re: DannyArends/0_installSoftware.sh @Olamzq commented on this gist.

________________________________ Dear Danny, Thank you for the video. I am trying to run step 3 (Remove duplicate reads using picard tools) of this lecture, but it's showing an error message at 1:30:00<https://www.youtube.com/watch?v=hWCDwLByxvs&t=5400s> of the video. I have tried to look for a solution online, but none is providing a solution to the problem. I would appreciate your help. Thank you. [Sun Jan 14 14:55:18<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53718s> CST 2024] Executing as ***@***.*** on Linux 6.1.0-17-amd64 amd64; OpenJDK 64-Bit Server VM 17.0.9+9-Debian-1deb12u1; Deflater: Intel; Inflater: Intel; Provider GCS is available; Picard version: Version:3.1.1-8-ga638ce92a-SNAPSHOT INFO 2024-01-14 14:55:18<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53718s> MarkDuplicates Start of doWork freeMemory: 38556984; totalMemory: 50331648; maxMemory: 4294967296 INFO 2024-01-14 14:55:18<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53718s> MarkDuplicates Reading input file and constructing read end information. INFO 2024-01-14 14:55:18<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53718s> MarkDuplicates Will retain up to 15561475 data points before spilling to disk. WARNING 2024-01-14 14:55:19<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53719s> AbstractOpticalDuplicateFinderCommandLineProgram A field field parsed out of a read name was expected to contain an integer and did not. Read name: SRR13978643.342872. Cause: String 'SRR13978643.342872' did not start with a parsable number. [Sun Jan 14 14:55:19<https://www.youtube.com/watch?v=hWCDwLByxvs&t=53719s> CST 2024] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 0.02 minutes. Runtime.totalMemory()=241172480 To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp Exception in thread "main" java.lang.NullPointerException: Cannot invoke "htsjdk.samtools.SAMReadGroupRecord.getReadGroupId()" because the return value of "htsjdk.samtools.SAMRecord.getReadGroup()" is null at picard.sam.markduplicates.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:558) at picard.sam.markduplicates.MarkDuplicates.doWork(MarkDuplicates.java:270) at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:280) at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:105) at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:115) 1 Error external process did not finish — Reply to this email directly, view it on GitHub<https://gist.github.com/DannyArends/04d87f5590090dfe0dc6b42e5e1bbe15#gistcomment-4830835> or unsubscribe<https://github.com/notifications/unsubscribe-auth/AAALWHWYRUNVWCBXLJIEHXTYORHRPBFKMF2HI4TJMJ2XIZLTSKBKK5TBNR2WLJDUOJ2WLJDOMFWWLO3UNBZGKYLEL5YGC4TUNFRWS4DBNZ2F6YLDORUXM2LUPGBKK5TBNR2WLJDHNFZXJJDOMFWWLK3UNBZGKYLEL52HS4DFVRZXKYTKMVRXIX3UPFYGLK2HNFZXIQ3PNVWWK3TUUZ2G64DJMNZZDAVEOR4XAZNEM5UXG5FFOZQWY5LFVEYTCOBXGYZDSMZZU52HE2LHM5SXFJTDOJSWC5DF>. You are receiving this email because you authored the thread. Triage notifications on the go with GitHub Mobile for iOS<https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android<https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>.

Hi, you managed to solve the error, I have the same problem, how did you solve it?

Unfortunately I wasn’t able to solve the problem and I have been busy with my other projects since then. I haven’t been able to take a look at it since then. I hope there is a solution soon Get Outlook for iOS<https://aka.ms/o0ukef>

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________________________________ From: Chris091089 ***@***.***> Sent: Thursday, March 14, 2024 3:00:54 PM To: Chris091089 ***@***.***> Cc: Comment ***@***.***> Subject: Re: DannyArends/0_installSoftware.sh @Chris091089 commented on this gist.

________________________________ Hi, you managed to solve the error, I have the same problem, how did you solve it? — Reply to this email directly, view it on GitHub<https://gist.github.com/DannyArends/04d87f5590090dfe0dc6b42e5e1bbe15#gistcomment-4988232> or unsubscribe<https://github.com/notifications/unsubscribe-auth/AWPMR7R7Q2Y4T3VGDZ6WJYTYYH6XNBFKMF2HI4TJMJ2XIZLTSKBKK5TBNR2WLJDUOJ2WLJDOMFWWLO3UNBZGKYLEL5YGC4TUNFRWS4DBNZ2F6YLDORUXM2LUPGBKK5TBNR2WLJDHNFZXJJDOMFWWLK3UNBZGKYLEL52HS4DFVRZXKYTKMVRXIX3UPFYGLK2HNFZXIQ3PNVWWK3TUUZ2G64DJMNZZDAVEOR4XAZNEM5UXG5FFOZQWY5LFVEYTCOBXGYZDSMZZU52HE2LHM5SXFJTDOJSWC5DF>. You are receiving this email because you commented on the thread. Triage notifications on the go with GitHub Mobile for iOS<https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android<https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>.

Thank you very much, the problem is essentially a matter of some SAMTOOLS updates, be careful, the version that did not work for me is:
3.1.1-16-g5b0b4c014-SNAPSHOT, the version that works is: 2.18.27-SNAPSHOT installed in docker taken from the following link: https://hub.docker.com/r/agrf/picard

Thanks for the support.

Thank you. I will take a look at it. Best, Olamzq Get Outlook for iOS<https://aka.ms/o0ukef>

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________________________________ From: Chris091089 ***@***.***> Sent: Friday, March 15, 2024 4:51:51 PM To: Chris091089 ***@***.***> Cc: Comment ***@***.***> Subject: Re: DannyArends/0_installSoftware.sh @Chris091089 commented on this gist.

________________________________ Thank you very much, the problem is essentially a matter of some SAMTOOLS updates, be careful, the version that did not work for me is: 3.1.1-16-g5b0b4c014-SNAPSHOT, the version that works is: 2.18.27-SNAPSHOT installed in docker taken from the following link: https://hub.docker.com/r/agrf/picard Thanks for the support. — Reply to this email directly, view it on GitHub<https://gist.github.com/DannyArends/04d87f5590090dfe0dc6b42e5e1bbe15#gistcomment-4989834> or unsubscribe<https://github.com/notifications/unsubscribe-auth/AWPMR7WRJLQ3P52IWNENHBLYYNUPPBFKMF2HI4TJMJ2XIZLTSKBKK5TBNR2WLJDUOJ2WLJDOMFWWLO3UNBZGKYLEL5YGC4TUNFRWS4DBNZ2F6YLDORUXM2LUPGBKK5TBNR2WLJDHNFZXJJDOMFWWLK3UNBZGKYLEL52HS4DFVRZXKYTKMVRXIX3UPFYGLK2HNFZXIQ3PNVWWK3TUUZ2G64DJMNZZDAVEOR4XAZNEM5UXG5FFOZQWY5LFVEYTCOBXGYZDSMZZU52HE2LHM5SXFJTDOJSWC5DF>. You are receiving this email because you commented on the thread. Triage notifications on the go with GitHub Mobile for iOS<https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android<https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>.

Hello, Could you please send me a short clip of how you fixed the problem. I would be very grateful. Thank you very much. Best regards, Olamide Sent from Outlook<http://aka.ms/weboutlook>

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________________________________ From: Chris091089 ***@***.***> Sent: Friday, March 15, 2024 4:51 PM To: Chris091089 ***@***.***> Cc: Comment ***@***.***> Subject: Re: DannyArends/0_installSoftware.sh @Chris091089 commented on this gist.

________________________________ Thank you very much, the problem is essentially a matter of some SAMTOOLS updates, be careful, the version that did not work for me is: 3.1.1-16-g5b0b4c014-SNAPSHOT, the version that works is: 2.18.27-SNAPSHOT installed in docker taken from the following link: https://hub.docker.com/r/agrf/picard Thanks for the support. — Reply to this email directly, view it on GitHub<https://gist.github.com/DannyArends/04d87f5590090dfe0dc6b42e5e1bbe15#gistcomment-4989834> or unsubscribe<https://github.com/notifications/unsubscribe-auth/AWPMR7WRJLQ3P52IWNENHBLYYNUPPBFKMF2HI4TJMJ2XIZLTSKBKK5TBNR2WLJDUOJ2WLJDOMFWWLO3UNBZGKYLEL5YGC4TUNFRWS4DBNZ2F6YLDORUXM2LUPGBKK5TBNR2WLJDHNFZXJJDOMFWWLK3UNBZGKYLEL52HS4DFVRZXKYTKMVRXIX3UPFYGLK2HNFZXIQ3PNVWWK3TUUZ2G64DJMNZZDAVEOR4XAZNEM5UXG5FFOZQWY5LFVEYTCOBXGYZDSMZZU52HE2LHM5SXFJTDOJSWC5DF>. You are receiving this email because you commented on the thread. Triage notifications on the go with GitHub Mobile for iOS<https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android<https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>.

@Chris091089 I am encountering the same error which you faced and resolve earlier. I tried to download picard version 2.18.27-SNAPSHOT from your shared link. But it ended up in the following error:
Using default tag: latest
Cannot connect to the Docker daemon at unix:///var/run/docker.sock. Is the docker daemon running?

did you encounter the same issue. If yes, please help. I would be more than thankful for this favour.

Dear all,

I have edited the 0_installSoftware.sh script to checkout the correct version of PICARD and STAR, this should fix the:

Exception in thread "main" java.lang.NullPointerException: Cannot invoke "htsjdk.samtools.SAMReadGroupRecord.getReadGroupId()" because the return value of "htsjdk.samtools.SAMRecord.getReadGroup()" is null

error encountered by some of you

Hi @DannyArends ,
I'm done with PICARD duplictate error by upgrading picard & java. And I'm already thankful to you for helping. But when I try to run this command ./igv.sh my IGV viewer is not opening. I wonder if you can help me to sort it. When I run command it shows this:
echo Using bundled JDK.
WARNING: package com.sun.java.swing.plaf.windows not in java.desktop
WARNING: package sun.awt.windows not in java.desktop
openjdk version "11.0.13" 2021-10-19
OpenJDK Runtime Environment Temurin-11.0.13+8 (build 11.0.13+8)
OpenJDK 64-Bit Server VM Temurin-11.0.13+8 (build 11.0.13+8, mixed mode)
INFO [Apr 22,2024 08:59] [Globals] Development mode is enabled
SEVERE [Apr 22,2024 08:59] [DefaultExceptionHandler] Unhandled exception
SEVERE [Apr 22,2024 08:59] [DefaultExceptionHandler] java.awt.AWTError: Can't connect to X11 window server using ':0' as the value of the DISPLAY variable.
at java.desktop/sun.awt.X11GraphicsEnvironment.initDisplay(Native Method)
at java.desktop/sun.awt.X11GraphicsEnvironment$1.run(Unknown Source)
at java.base/java.security.AccessController.doPrivileged(Native Method)
at java.desktop/sun.awt.X11GraphicsEnvironment.(Unknown Source)
at java.base/java.lang.Class.forName0(Native Method)
at java.base/java.lang.Class.forName(Unknown Source)
at java.desktop/java.awt.GraphicsEnvironment$LocalGE.createGE(Unknown Source)
at java.desktop/java.awt.GraphicsEnvironment$LocalGE.(Unknown Source)
at java.desktop/java.awt.GraphicsEnvironment.getLocalGraphicsEnvironment(Unknown Source)
at java.desktop/sun.awt.X11.XToolkit.(Unknown Source)
at java.base/java.lang.Class.forName0(Native Method)
at java.base/java.lang.Class.forName(Unknown Source)
at java.desktop/java.awt.Toolkit$2.run(Unknown Source)
at java.desktop/java.awt.Toolkit$2.run(Unknown Source)
at java.base/java.security.AccessController.doPrivileged(Native Method)
at java.desktop/java.awt.Toolkit.getDefaultToolkit(Unknown Source)
at java.desktop/java.awt.Toolkit.getEventQueue(Unknown Source)
at java.desktop/java.awt.EventQueue.invokeLater(Unknown Source)
at java.desktop/javax.swing.SwingUtilities.invokeLater(Unknown Source)
at org.igv/org.broad.igv.ui.Main.main(Main.java:124)

@aqibafarman this is an issue with the X11 display under Linux not being setup properly and happens when you use a linux which runs in headless mode. E.g. while logging into a server or cluster remotely or when are you using WSL2 instead of a virtual box. The easiest is to transfer the files to windows and use the IGV under window.