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Old 09-18-2013, 05:08 PM   #1
AnushaC
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Default bam-load

Hi Everyone ,
I am trying to use bam-load function to convert an SRA file to bam file .



bam-load.2.1.13 [options] <bam-file>

Summary:
Load a BAM formatted data file

Example:
bam-load.2.1.13 -o /tmp/SRZ123456 -k analysis.bam.cfg 123456.bam
Can anyone can explain what options . What is exactly analysis.bam.cfg
what input's it takes and what is output in detail.

Thanks,
Anusha.Ch
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Old 09-18-2013, 07:09 PM   #2
crazyhottommy
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SRA file usually contains unmapped reads, you need to use the fastq-dump in the sra-tool kit to dump it to fastq, then you map it with bowtie which gives you a sam file, you then can use samtools to convert sam to bam.
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Old 09-20-2013, 01:26 PM   #3
AnushaC
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Default Using Bowtie to map fastq to sam file format

Hi Everyone ,
Can anyone give examples of using bowtie to map from fastq to sam format .

I did try all the options it is not working for me


/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2 -1 /raid/development/anusha/SRR203400.fastq --U - -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19|-U /-1 ./SRR203400.fastq -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19 |-U </raid/software/development/SRR203400.fastq> -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19| -u/ -1 /raid/development/anusha/SRR203400.fastq -S /raid/development/anusha/SRR2034001.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19 | -u 1 /raid/development/anusha/SRR203400.fastq -S /raid/development/anusha/SRR2034001.sam

bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]
can anyone what is option u ,what it takes with detailed example

my errors are always like this
Error: Must specify at least one read input with -U/-1/-2
bowtie2-align exited with value 1

Thanks,
Anusha.Ch
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Old 09-21-2013, 12:20 AM   #4
dpryan
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Default

Quote:
Originally Posted by AnushaC View Post
Hi Everyone ,
Can anyone give examples of using bowtie to map from fastq to sam format .

I did try all the options it is not working for me


/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2 -1 /raid/development/anusha/SRR203400.fastq --U - -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19|-U /-1 ./SRR203400.fastq -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19 |-U </raid/software/development/SRR203400.fastq> -S /raid/development/anusha/SRR203400.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19| -u/ -1 /raid/development/anusha/SRR203400.fastq -S /raid/development/anusha/SRR2034001.sam

/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2/hg19 | -u 1 /raid/development/anusha/SRR203400.fastq -S /raid/development/anusha/SRR2034001.sam

bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]
can anyone what is option u ,what it takes with detailed example

my errors are always like this
Error: Must specify at least one read input with -U/-1/-2
bowtie2-align exited with value 1

Thanks,
Anusha.Ch
Firstly, you should start a new thread next time. Secondly, the error message is because you misspecify the bowtie2 command. "{-1 <m1> -2 <m2> | -U <r>}" means you either use both -1 and -2 (i.e., you have paired-end reads) or you use only -U (i.e. you have single end reads). A proper command would be more like:
Code:
/raid/software/src/bowtie2 -q -x /raid/references-and-indexes/hg19/bowtie2 -U /raid/development/anusha/SRR203400.fastq -S /raid/development/anusha/SRR203400.sam
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Old 09-21-2013, 10:18 AM   #5
AnushaC
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Default

Thanks for reply ,
It is working


Thanks,
Anusha.Ch
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Old 09-24-2013, 04:26 PM   #6
AnushaC
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Default

Hi Everyone ,
First of all I am sorry that I am not using new thread . I want to use bowtie default 0.12.7 and i want to use option -n 3 ,- v3 and -k 2 .
opment/anusha/SRR203400.sam
Could not locate a Bowtie index corresponding to basename "s"
Command: bowtie -n 3 -v 3 -k 2 --sam s /raid/references-and-indexes/hg19/bowtie-indexes /raid/development/anusha/SRR203400.fastq
anusha@cn1:/raid/development/anusha> bowtie -n 3 -v 3 -k 2 --sam -c /raid/references-and-indexes/hg19/bowtie-indexes /raid/development/anusha/SRR203400.fastq > /raid/development/anusha/SRR203400.sam
Could not locate a Bowtie index corresponding to basename "/raid/references-and-indexes/hg19/bowtie-indexes"
Command: bowtie -n 3 -v 3 -k 2 --sam -c /raid/references-and-indexes/hg19/bowtie-indexes /raid/development/anusha/SRR203400.fastq
anusha@cn1:/raid/development/anusha>

I did not understand what is going wrong

this is my reference index location and files

cd /raid/references-and-indexes/hg19/bowtie-indexes
anusha@cn1:/raid/references-and-indexes/hg19/bowtie-indexes> ls
chrM-tRNA-rRNA-genes_c.1.ebwt chrM-tRNA-rRNA-genes_c.4.ebwt chrM-tRNA-rRNA-genes.fa hg19.3.ebwt hg19_c.2.ebwt hg19_c.rev.1.ebwt hg19.rev.2.ebwt
chrM-tRNA-rRNA-genes_c.2.ebwt chrM-tRNA-rRNA-genes_c.rev.1.ebwt hg19.1.ebwt hg19.4.ebwt hg19_c.3.ebwt hg19_c.rev.2.ebwt
chrM-tRNA-rRNA-genes_c.3.ebwt chrM-tRNA-rRNA-genes_c.rev.2.ebwt hg19.2.ebwt hg19_c.1.ebwt hg19_c.4.ebwt hg19.rev.1.ebwt
anusha@cn1:/raid/references-and-indexes/hg19/bowtie-indexes>

can anybody explain in detail how to use bowtie with clear example


Thanks very much ,
Anusha.Ch
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Old 09-24-2013, 11:19 PM   #7
dpryan
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Code:
bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq > /raid/development/anusha/SRR203400.sam
You can't use both -n and -v, they're mutually exclusive.
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Old 09-26-2013, 02:19 PM   #8
AnushaC
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Default Loading the bam data to IGV

Hi Everyone ,
Can any one can explain me in detail step by step how to upload your bam data to IGV . I have my bam file which is sorted and indexed.

Thanks In Advance ,
Anusha.ch
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Old 09-26-2013, 02:33 PM   #9
vivek_
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If your bam file is really large its not very advisable to open it with IGV, you'd rather use Samtools to create a subset of the BAM file for the regions you need, sort and index it and open it with IGV.

Otherwise, its just as simple as File->Open->Point to your bam in directory, change the reference genome based on your needs from the drop down menu. If your genome is not already there, you need to import it, which is slightly complicated.
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Old 09-26-2013, 02:37 PM   #10
AnushaC
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Default Opening file with IGV

Hi ,
I have my bam file sorted and indexed that is bam and bai files can anyone explain how to open those file with IGV in detail every step very clearly


Thanks,
Anusha.Ch
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Old 09-27-2013, 02:50 AM   #11
dpryan
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Quote:
Originally Posted by AnushaC View Post
Hi ,
I have my bam file sorted and indexed that is bam and bai files can anyone explain how to open those file with IGV in detail every step very clearly


Thanks,
Anusha.Ch
Click on "File" and then "Load", just like opening anything else. Have a read through the IGV user guide.
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Old 09-27-2013, 08:51 AM   #12
AnushaC
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Default Trying making files HTMl

anusha@cn1:/raid/development/anusha/IGV> chmod 755 SRR203400.bowtieold.bam
anusha@cn1:/raid/development/anusha/IGV> chmod 755 SRR203400.sort.bam.bam
anusha@cn1:/raid/development/anusha/IGV> chmod 755 SRR203400.sort.bam.bam.bai
anusha@cn1:/raid/development/anusha/IGV> ln -s /raid/development/anusha/IGV/SRR203400.bowtieold.bam /raid/development/anusha/IGV/SRR203400.bowtieold.bam
ln: failed to create symbolic link ‘/raid/development/anusha/IGV/SRR203400.bowtieold.bam’: File exists
anusha@cn1:/raid/development/anusha/IGV> cd IGV
anusha@cn1:/raid/development/anusha/IGV/IGV> ls
IGV SRR203400.bowtieold.bam SRR203400.sort.bam.bam SRR203400.sort.bam.bam.bai
anusha@cn1:/raid/development/anusha/IGV/IGV> cd IGV
anusha@cn1:/raid/development/anusha/IGV/IGV/IGV> ls
IGV SRR203400.bowtieold.bam SRR203400.sort.bam.bam SRR203400.sort.bam.bam.bai
anusha@cn1:/raid/development/anusha/IGV/IGV/IGV> ls
IGV SRR203400.bowtieold.bam SRR203400.sort.bam.bam SRR203400.sort.bam.bam.bai
anusha@cn1:/raid/development/anusha/IGV/IGV/IGV> cd ..
anusha@cn1:/raid/development/anusha/IGV/IGV> cd ..
anusha@cn1:/raid/development/anusha/IGV> ls
IGV SRR203400.bowtieold.bam SRR203400.sort.bam.bam SRR203400.sort.bam.bam.bai
anusha@cn1:/raid/development/anusha/IGV> pwd
/raid/development/anusha/IGV
anusha@cn1:/raid/development/anusha/IGV> cd ..
anusha@cn1:/raid/development/anusha> ln -s /raid/development/anusha/SRR203400.bowtieold.bam /raid/development/anusha/SRR203400.bowtieold.bam
ln: failed to create symbolic link ‘/raid/development/anusha/SRR203400.bowtieold.bam’: File exists
anusha@cn1:/raid/development/anusha> Write failed: Broken pipe
Anushas-MacBook-Air:~ AnuSuri$ ssh anusha@flc.ucsd.edu
Password:
Last login: Fri Sep 27 09:44:53 2013 from 172.21.164.246

#################################
Personal compute nodes : cn1-cn3
Scheduled compute nodes: cn4-cn19
#################################

anusha@hn:~> ls
50M-baits_bait.interval_list1 ANUSHA_PROJECT humanV4-baits_hg19_lite.interval_list _mappedHuman_nameSorted.bam ncbi_error_report.xml shell_script_name.sh
50M-targets_hg19_lite.interval_list1 bin humanV4-targets_hg19_lite.interval_list _mappedMouse_nameSorted.bam public_html
anusha@hn:~> cd public_html
anusha@hn:~/public_html> ls
luad_exome.bed luad_exome.copy.bed samplecov3.bedgraph samplecovhg19.chr.bedgraph
anusha@hn:~/public_html> cd ...
-bash: cd: ...: No such file or directory
anusha@hn:~/public_html> cd ..
anusha@hn:~> ln -s /raid/development/anusha/SRR203400.bowtieold.bam /raid/development/anusha/SRR203400.bowtieold.bam
ln: failed to create symbolic link `/raid/development/anusha/SRR203400.bowtieold.bam': File exists
anusha@hn:~>

Hi I did try to make my files HTML . I put all my files in a folder and applied command but i can't see public folder but if i try for individual files it showing file already there . Can anyone explain me what is happening and also i did not understand what is meant by click and load can anybody explain me in detail how to do IGV?
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Old 09-27-2013, 09:58 AM   #13
dpryan
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Quote:
Originally Posted by AnushaC View Post
anusha@cn1:/raid/development/anusha/IGV> ln -s /raid/development/anusha/IGV/SRR203400.bowtieold.bam /raid/development/anusha/IGV/SRR203400.bowtieold.bam
Creating a symbolic link from a file to itself is a rather odd thing to try to do. It will never work and I haven't a clue what you're trying to accomplish by even trying.

Quote:
Originally Posted by AnushaC View Post
anusha@cn1:/raid/development/anusha> ln -s /raid/development/anusha/SRR203400.bowtieold.bam /raid/development/anusha/SRR203400.bowtieold.bam
Again, this will never work and I have no clue why you're trying to do this. This has nothing to do with making an HTML page, as per your title.

So, onto the second computer...

Quote:
Originally Posted by AnushaC View Post
anusha@hn:~> ln -s /raid/development/anusha/SRR203400.bowtieold.bam /raid/development/anusha/SRR203400.bowtieold.bam
See above.

Quote:
Originally Posted by AnushaC View Post
Can anyone explain me what is happening and also i did not understand what is meant by click and load can anybody explain me in detail how to do IGV?
Start IGV (if you have it installed properly, just type IGV from the command line. You will then have a GUI and the instructions of "Click 'File' and then 'Load'" will make more sense. You would do well to get some help locally from someone familiar with using linux and other unix like systems as I suspect most of your issues will boil down to "basic"* computer usage.

*Well, as basic as using *nix-like systems can ever be considered.
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Old 09-27-2013, 12:02 PM   #14
AnushaC
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Default fastq to sam want to use -F options to know only the read that are aligned to genome

Hi Everyone ,
can anyone can explain me how to use -F option for sam output to know the only the reads that are aligned to genome .



anusha@cn1:/raid/development/anusha> bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq > samtools view -S F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
Extra parameter(s) specified: "F", "/raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam"
Command: bowtie -v 3 -k 2 --sam -S /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq view F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
anusha@cn1:/raid/development/anusha> bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fast -F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
Command: bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fast -F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
anusha@cn1:/raid/development/anusha>
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Old 09-27-2013, 04:16 PM   #15
AnushaC
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Default Checking for SNP in IGV

Hi Everybody ,
Can anyone explain me how to look for the SNP's in IGV browser


Thanks,
Anusha.Ch
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Old 09-28-2013, 06:07 AM   #16
dpryan
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Quote:
Originally Posted by AnushaC View Post
anusha@cn1:/raid/development/anusha> bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq > samtools view -S F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
Extra parameter(s) specified: "F", "/raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam"
Command: bowtie -v 3 -k 2 --sam -S /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq view F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
anusha@cn1:/raid/development/anusha> bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fast -F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
Command: bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fast -F /raid/development/anusha/SRR203400.bowtieold.flagtrnc.sam
anusha@cn1:/raid/development/anusha>
I assume you mean:
Code:
bowtie -v 3 -k 2 --sam /raid/references-and-indexes/hg19/bowtie-indexes/hg19 /raid/development/anusha/SRR203400.fastq | samtools view -S -F 0x4 -o /raid/development/anusha/SRR203400.bowtieold.flagtrnc.bam -
Or something like that, which will write to a BAM file and ignore unaligned reads.
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Old 09-28-2013, 06:11 AM   #17
dpryan
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Quote:
Originally Posted by AnushaC View Post
Hi Everybody ,
Can anyone explain me how to look for the SNP's in IGV browser


Thanks,
Anusha.Ch
I assume you mean "look at SNPs", since it would take forever to manually look for them in the whole genome. You should be able to answer that question yourself (hint, what should a SNP look like?).
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Old 10-01-2013, 02:13 PM   #18
AnushaC
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Fastq dump and finding the contiguous reads covered by at least one read

Hi Everyone ,
I have couple of questions . Can anybody explain me in detail answers

1) When u are using fast dump if u want to output to specific place where you want what option in fastq dunpl wil give this.

fastq-dump [options] [ -A ]
fastq-dump [options]
what is the accession in the option what for it used because if i gave without option A it worked. I am using shell script to automate my process i want the output to the place i wanted .
I used above shell script it worked
#!/bin/bash
# generating a fastq file using fastq dump

fastq-dump $2 $1
but while running it came like this why ?


anusha@cn1:/raid/development/anusha/python_test/shelltest> ./SRAtoBAM.sh /raid/development/anusha/python_test/shelltest/SRR203400.sra /raid/development/anusha/python_test/shelltest/SRR203400.fastq
2013-10-01T22:02:53 fastq-dump.2.1.12 err: table not found while opening manager within database module - failed to open '/raid/development/anusha/python_test/shelltest/SRR203400.fastq'
Written 16472407 spots for /raid/development/anusha/python_test/shelltest/SRR203400.sra
Written 16472407 spots total

2) what tools will give " contiguous reads covered by at least one read "


Thanks so much in advance

Thanks,
Anusha.Ch
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