SPRINT is Exiting

[HughCruiseJr]

Thank you for building SPRINT, I read about this and got fascinated about it and how you developed it!!
I am novice to the field of bioinformatics and in my junior year, please help me with this.

I am working with RNA seq data and I have to find particular editing in this data also I would be using SPRINT's A2I script, now I have quality checked fastq.gz files and I have annotated them and made SORTEDBAM files out of it so no need to gunzip. Now when I am running SPRINT with satifying all pre-requisite (python2, Samtools etc) this being my code

"python2 sprint_from_bam.py --rp $HOME/Output/simpleRepeat.txt --bam $HOME/Output/STAR_Output/Aligned_B1_53632Aligned.sortedByCoord.out_.bam --genome $HOME/Output/GRCh38.primary_assembly.genome.fa --out $HOME/Output/Sprint_B1_53632 /usr/bin/samtools"

my SPRINT is exiting without no error and nothing is happening. I am not sure what I am missing.

Thank you for your response in advance.

Best,
A

[jumphone]

Thanks for using sprint,

1. plz try:

python2 sprint_from_bam.py --rp $HOME/Output/simpleRepeat.txt $HOME/Output/STAR_Output/Aligned_B1_53632Aligned.sortedByCoord.out_.bam $HOME/Output/GRCh38.primary_assembly.genome.fa $HOME/Output/Sprint_B1_53632 /usr/bin/samtools

plz keep "-rp" and remove "--bam", "--genome", and "-out".

2. plz download repeat file from https://sourceforge.net/projects/sprintpy/files/dbRES/

You can find a "dbrep.zip" there.

Feng

[HughCruiseJr]

Thank you so much for your reply, my repeat file is from UCSC specifically for HG38, cause when I did the annotations I used GTF file for HG38? also the dbrep, I am interested in human, but it has so many txt files, would I need to choose one from them or like how I need to use them?

also I have python 2.7 but as i was searching more in depth, I saw that my pysam is having some problem, do you think that would do any problem?

Thank you so much again

[HughCruiseJr]

I ran the suggestions that you gave earlier, "a7@bios-76-1:~/Output/SPRINT$ python2 sprint_from_bam.py --rp $HOME/Output/Human/SRR2040582.regular.txt $HOME/Output/STAR_Output/Aligned_B1_53632Aligned.sortedByCoord.out_.bam $HOME/Output/GRCh38.primary_assembly.genome.fa $HOME/Output/Sprint_B1_53632 /usr/bin/samtools" but still its exiting. I am little overwhelmed.

Thank you again in advance for your help.

Best,
A

[jumphone]

1 “-rp”, not "--rp".

2 “dbrep.zip”, not “dbres.zip”.

3 I highly recommend that you run SPRINT from the FQ file, rather than sprint_from_bam, because different aligners may produce different “types” of BAM files. Running "sprint" from the FQ file is preferable.

[HughCruiseJr]

Thank you doer your response, my program ran, but all the resulting files were 0KB, only aligned SAM and genome_all_zz.dedup are having data. I got the BAM files from STAR and annotated them in aligned manner only but still I am not getting my results.

Simultaneously, I am using sprint prepare and then sprint main, but while running sprint prepare again its exiting without any results

"a7@bios-76-1:~/Output/SPRINT$ python2 sprint_prepare.py -t $HOME/Output/gencode.v29.annotation.gtf $HOME/Output/GRCh38.primary_assembly.genome.fa /usr/bin/bwa"

Thank you again!

Best

[jumphone]

1. you can directly use the binary version of sprint: https://github.com/jumphone/SPRINT/tree/master/bin

2. you can download the bwa and samtools from https://github.com/jumphone/SPRINT/tree/master/samtools_and_bwa

3. make them execuable:
   chmod 777 sprint, chmod 777 bwa, chmod 777 samtools

4. please download the FA and GTF from:
   https://pan.baidu.com/s/1R6N944QkZhvcvunIigVcXg?pwd=jump

5. ./sprint prepare -t GTF FA BWA

[HughCruiseJr]

Thank you for your reply feng.
Can you please let me know any other way to download the files instead of Pan.Baidu, cause it is taking forever to download the files. Can I download from here, https://www.gencodegenes.org/human/release_39.html, & https://hgdownload.soe.ucsc.edu/goldenpath/hg38/bigZips/ ? Also in your 5th step, it is the same way that I wrote in my previous comment, I see no difference, can you please let me know what step I am doing wrong? Thank you again!

[jumphone]

1. Maybe the gtf and the fa are not matched. Even for GRCH38, there are a lot of ways to record the name of chromesomes (e.g. CHR1, chr1, Chr1, 1)

2. "sprint prepare" will output index files in the folder of your FA file. Could you plz check the folder of your FA file?

[HughCruiseJr]

Thank you for your reply, I was able to retrieve files, and I have them all in same directory to avoid confusion. This is the code that I am writing and its exiting without even giving the basic information which I was able to see previously with Sprint_from_bam.py (sprint prepare [options] reference_genome(.fa) bwa_path"
options: -t transcript_annotation(.gtf) #Optional
sprint prepare -t hg19.gtf hg19.fa ./bwa-0.7.12/bwa")

"a7@bios-76-1:~/Output/SPRINT/sprint$ python2 sprint_prepare.py -t gencode.v39.annotation.gtf hg38.fa /usr/bin/bwa"

Everything is in the manner script required.

Looking forward for your esteemed reply.

[jumphone]

Thank you for your reply, I was able to retrieve files, and I have them all in same directory to avoid confusion. This is the code that I am writing and its exiting without even giving the basic information which I was able to see previously with Sprint_from_bam.py (sprint prepare [options] reference_genome(.fa) bwa_path" options: -t transcript_annotation(.gtf) #Optional sprint prepare -t hg19.gtf hg19.fa ./bwa-0.7.12/bwa")

"a7@bios-76-1:~/Output/SPRINT/sprint$ python2 sprint_prepare.py -t gencode.v39.annotation.gtf hg38.fa /usr/bin/bwa"

Everything is in the manner script required.

Looking forward for your esteemed reply.

Hi, there are two ways to run SPRINT (the lastest version on github):

1. python2 run.py prepare or main

2. directly use the binary version "sprint"

Plz try: python2 run.py prepare -t GTF FA BWA

You can check the python2 code of each "py" file. I move the "main" function to the run.py in the lastest version.

Here is my code:

GTF=/home/database/annotation/hg19/Homo_sapiens.GRCh37.75.chr.gtf
REF=/home/database/pipeline/sprint/try_prepare/hg19.fa
BWA=/home/database/pipeline/sprint/bin/bwa

python2 /home/database/pipeline/sprint/src/SPRINT-master/run.py prepare -t $GTF $REF $BWA

It will report nothing when running correctly, but you can check the folder of your .fa file. The first generated file is "hg19.fa.mskAG.fa" for my code.

[HughCruiseJr]

Thank you so much Feng!
I was able to prepare, and then I ran main function but after running for 10-12 minutes its stopping, seems like not able to find some files I don't understand why that's happening.

"a7@bios-76-1:~/Output$ python2 /home/pandea7/Output/SPRINT/run.py main -p 6 -1 /home/pandea7/Output/HPAP-001/HPAP001B1_53632.fastq -rp /home/pandea7/Output/SPRINT/hg38_repeat.txt $REF /home/pandea7/Output/SPRINT/Output_Sprint $BWA $samtools"

I checked all the files, they are at there respective locations.

Looking forward to your esteemed reply.

Best,

[jumphone]

You need to use the bwa and samtools in the correct version.

You can directly download them from: https://github.com/jumphone/SPRINT/tree/master/samtools_and_bwa

[HughCruiseJr]

Dear Feng,
Thank you for your response, samtools and BWA are already downloaded with correct version on my system, still facing the same problem as mentioned above. This is the code that I am writing

"python2 /home/pandea7/Output/SPRINT/run.py main -p 6 -1 /home/pandea7/Output/HPAP-001/HPAP001B1_53632.fastq -rp /home/pandea7/Output/SPRINT/hg38_repeat.txt $REF /home/pandea7/Output/SPRINT/Output_Sprint $BWA $samtools"

Problem is same as above, I realized why you ask me to check my BWA & samtools, I read that samtools is unable to open the file "Output_Sprint//tmp//transcript_mskTC/all.bam" : No such file or directory" in directory we have "transcript_mskTC" but inside of that we just have file "name_read1.bam & name_read1.sam" other that that we don't have all.bam.

I already Chmod -R 777 samtools&bwa, but problem persist, I checked the files too. I am very clueless.

Thank you so much in advance for your patience and explanations.

Best,

[jumphone]

Please send the whole error report to "jumphone@163.com"

If you can see the help document of "samtools sort" in the error report, you use the wrong version of samtools.

[jumphone]

Dear Feng, Thank you for your response, samtools and BWA are already downloaded with correct version on my system, still facing the same problem as mentioned above. This is the code that I am writing

"python2 /home/pandea7/Output/SPRINT/run.py main -p 6 -1 /home/pandea7/Output/HPAP-001/HPAP001B1_53632.fastq -rp /home/pandea7/Output/SPRINT/hg38_repeat.txt $REF /home/pandea7/Output/SPRINT/Output_Sprint $BWA $samtools"

Problem is same as above, I realized why you ask me to check my BWA & samtools, I read that samtools is unable to open the file "Output_Sprint//tmp//transcript_mskTC/all.bam" : No such file or directory" in directory we have "transcript_mskTC" but inside of that we just have file "name_read1.bam & name_read1.sam" other that that we don't have all.bam.

I already Chmod -R 777 samtools&bwa, but problem persist, I checked the files too. I am very clueless.

Thank you so much in advance for your patience and explanations.

Best,

Hi,

I've checked your error report. And I've sucessfully used "run.py" to get RESs by myself this afternoon.

plz:

1. remove previous result folder (rm -rf output)

2. use the commands in the following format:

REF=/home/database/reference/hg19/hg19.fa
REPEAT=/home/database/dbrep/hg19_repeat.txt
FQ1=./data/SRR388226_top1m_1.fastq
BWA=./bin/bwa
SAMTOOLS=./bin/samtools

python2 ./src/SPRINT-master/run.py main -rp $REPEAT -c 6 -p 6 -1 $FQ1 $REF $FQ1_sprint $BWA $SAMTOOLS

3. plz double check the SAMTOOLS. Your error report records the "help document" of "samtools sort". SPRINT uses an old version of samtools. And the old samtools uses a different way to assign the output of "samtools sort".

Best,
Feng