[GenoMax]

At this point try following.

$ cd desktop/bioinformatics/bow-tie/bowtie2-2.2.3

$ bowtie2 -x example/index/lambda_virus -1 example/reads/reads_1.fq -2 example/reads/reads_2.fq -S eg2.sam

You really need to go back and understand the concept of relative paths, working directories (this info is included in the Korf lab unix course I had pointed you to).

[zillur]

Thank you very much. Here is my bash profile. I have tried. But.....May be I am missing something....

upr189081:~ ZILLURRAHMAN$ open ~/.bash_profile
upr189081:~ ZILLURRAHMAN$ open ~/.bash_profile
upr189081:~ ZILLURRAHMAN$ cd desktop/bioinformatics/bow-tie/bowtie2-2.2.3/example/
upr189081:example ZILLURRAHMAN$ $BT2_HOME/bowtie2 -x $BT2_HOME/example/index/lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam
-bash: /bowtie2: No such file or directory
upr189081:example ZILLURRAHMAN$ cat ~/.bash_profile
export PATH="/Users/ZILLURRAHMAN/Desktop/Bioinformatics/Bow-tie/bowtie2-2.2.3/:$PATH"
export PATH=“users/ZILLURRAHMAN/desktop/bioinformatics/bow-tie/bowtie2-2.2.3/BT2_HOME:$PATH”
upr189081:example ZILLURRAHMAN$ cd
upr189081:~ ZILLURRAHMAN$ open ~/.bash_profile
upr189081:~ ZILLURRAHMAN$ $BT2_HOME/bowtie2 -x $BT2_HOME/example/index/lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam
-bash: /bowtie2: No such file or directory
upr189081:~ ZILLURRAHMAN$ cat ~/.bash_profile
export PATH="/Users/ZILLURRAHMAN/Desktop/Bioinformatics/Bow-tie/bowtie2-2.2.3/:$PATH"
export PATH=“users/ZILLURRAHMAN/desktop/bioinformatics/bow-tie/bowtie2-2.2.3/BT2_HOME:$PATH”
$ export BT2_HOME=/path_to/desktop/bioinformatics/bow-tie/bowtie2-2.2.3
upr189081:~ ZILLURRAHMAN$

[GenoMax]

$BT2_HOME designates a variable that you need to set, before you can use it in a command line.

$ export BT2_HOME=/path_to/desktop/bioinformatics/bow-tie/bowtie2-2.2.3

Replace the path_to part with real full path.

[zillur]

I don't know. Here is my directory.
upr189081:~ ZILLURRAHMAN$ cd desktop/bioinformatics/bow-tie/bowtie2-2.2.3/example/

[GenoMax]

What is $BT2_HOME set to? Is bowtie2 in $BT2_HOME directory?

[zillur]

Thank you very much. I am using these following command lines. But:-
upr189081:reference ZILLURRAHMAN$ $BT2_HOME/bowtie2 -x $BT2_HOME/example/index/lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam
-bash: /bowtie2: No such file or directory
upr189081:reference ZILLURRAHMAN$

What should I do now?

[GenoMax]

What next? I aligned the example lambda virus. Now I want to convert the sam file into bam file and then using flagstat I have to assess the quality of the alignments.

Regards
Zillur

http://bowtie-bio.sourceforge.net/bo...ols-downstream

[zillur]

Thank you very much. Yes, I think its working now.

Mds-MacBook-Pro:samtools ZILLURRAHMAN$ samtools-1.1/samtools

Program: samtools (Tools for alignments in the SAM format)
Version: 1.1 (using htslib 1.1)

Usage: samtools <command> [options]

Commands:
-- indexing
faidx index/extract FASTA
index index alignment
-- editing
calmd recalculate MD/NM tags and '=' bases
fixmate fix mate information
reheader replace BAM header
rmdup remove PCR duplicates
targetcut cut fosmid regions (for fosmid pool only)
-- file operations
bamshuf shuffle and group alignments by name
cat concatenate BAMs
merge merge sorted alignments
mpileup multi-way pileup
sort sort alignment file
split splits a file by read group
bam2fq converts a BAM to a FASTQ
-- stats
bedcov read depth per BED region
depth compute the depth
flagstat simple stats
idxstats BAM index stats
phase phase heterozygotes
stats generate stats (former bamcheck)
-- viewing
flags explain BAM flags
tview text alignment viewer
view SAM<->BAM<->CRAM conversion

Mds-MacBook-Pro:samtools ZILLURRAHMAN$
What next? I aligned the example lambda virus. Now I want to convert the sam file into bam file and then using flagstat I have to assess the quality of the alignments.

Regards
Zillur

[GenoMax]

Looks like the compilation is done. Can you run the samtools executable like so

$ samtools-1.1/samtools

That should print some help messages.

[zillur]

Thank you very much. I have typed "make" in the directory. Now my files are

Mds-MacBook-Pro:samtools-1.1 ZILLURRAHMAN$ ls
AUTHORS bam_plbuf.c faidx.c
ChangeLog.old bam_plbuf.h faidx.o
INSTALL bam_plbuf.o htslib-1.1
LICENSE bam_plcmd.c kaln.c
Makefile bam_plcmd.o kaln.h
Makefile.mingw bam_reheader.c kaln.o
NEWS bam_reheader.o kprobaln.c
README bam_rmdup.c kprobaln.h
bam.c bam_rmdup.o kprobaln.o
bam.h bam_rmdupse.c libbam.a
bam.o bam_rmdupse.o libhts.a
bam2bcf.c bam_sort.c misc
bam2bcf.h bam_sort.o padding.c
bam2bcf.o bam_split.c padding.o
bam2bcf_indel.c bam_split.o phase.c
bam2bcf_indel.o bam_stat.c phase.o
bam2depth.c bam_stat.o sam.c
bam2depth.o bam_tview.c sam.h
bam_aux.c bam_tview.h sam.o
bam_aux.o bam_tview.o sam_header.c
bam_cat.c bam_tview_curses.c sam_header.h
bam_cat.o bam_tview_curses.o sam_header.o
bam_color.c bam_tview_html.c sam_view.c
bam_color.o bam_tview_html.o sam_view.o
bam_endian.h bamshuf.c sample.c
bam_flags.c bamshuf.o sample.h
bam_flags.o bamtk.c sample.o
bam_import.c bamtk.o samtools
bam_import.o bedcov.c samtools.1
bam_index.c bedcov.o samtools.h
bam_index.o bedidx.c stats.c
bam_lpileup.c bedidx.o stats.o
bam_lpileup.h cut_target.c stats_isize.c
bam_lpileup.o cut_target.o stats_isize.h
bam_mate.c errmod.c stats_isize.o
bam_mate.o errmod.h test
bam_md.c errmod.o version.h
bam_md.o examples win32
Mds-MacBook-Pro:samtools-1.1 ZILLURRAHMAN$ make
make: Nothing to be done for `all'.

Is it compiled now? What should i do next?

Regards
Zillur

[GenoMax]

Samtools needs to be compiled. Have you done a "make" in the samtools directory? Does not look like you have based on the listing.

[zillur]

Thank you very much. Now after making the paths for indexing, build, align I can align bowtie2 files. I aligned the example lambda virus. Now I want to convert the sam file into bam file and then using flagstat I have to assess the quality of the alignments. I have downloaded samtools from

http://sourceforge.net/projects/samtools/files/latest/download?source=files

Now do I need to make path for the executables using such command lines
(export SAMTOOLS_HOME=/Users/ecerami/libraries/samtools-0.1.19
export PATH=$SAMTOOLS_HOME:$PATH
export PATH=$SAMTOOLS_HOME/bcftools/:$PATH
export PATH=$SAMTOOLS_HOME/misc/:$PATH)

There are too many files. Which are executables? If all are executables do I need to make path for all of these?

Regards
Zillur

Here is my files.
AUTHORS
bam_aux.c
bam_cat.c
bam_color.c
bam_endian.h
bam_flags.c
bam_import.c
bam_index.c
bam_lpileup.c
bam_lpileup.h
bam_mate.c
bam_md.c
bam_plbuf.c
bam_plbuf.h
bam_plcmd.c
bam_reheader.c
bam_rmdup.c
bam_rmdupse.c
bam_sort.c
bam_split.c
bam_stat.c
bam_tview_curses.c
bam_tview_html.c
bam_tview.c
bam_tview.h
bam.c
bam.h
bam2bcf_indel.c
bam2bcf.c
bam2bcf.h
bam2depth.c
bamshuf.c
bamtk.c
bedcov.c
bedidx.c
ChangeLog.old
cut_target.c
errmod.c
errmod.h
examples
faidx.c
htslib-1.1
INSTALL
kaln.c
kaln.h
kprobaln.c
kprobaln.h
LICENSE
Makefile
Makefile.mingw
misc
NEWS
padding.c
phase.c
README
sam_header.c
sam_header.h
sam_view.c
sam.c
sam.h
sample.c
sample.h
samtools.1
samtools.h
stats_isize.c
stats_isize.h
stats.c

[GenoMax]

What other contents are there in nohup.out file?

Are you following the steps in the bowtie tutorial here: http://bowtie-bio.sourceforge.net/bo...-phage-example

[zillur]

Thank you very much. Now can you tell me in which point I am wrong?
Here is my files and commands.


Mds-MacBook-Pro:~ ZILLURRAHMAN$ cd desktop/bioinformatics
Mds-MacBook-Pro:bioinformatics ZILLURRAHMAN$ cd bow-tie
Mds-MacBook-Pro:bow-tie ZILLURRAHMAN$ cd bowtie2-2.2.3
Mds-MacBook-Pro:bowtie2-2.2.3 ZILLURRAHMAN$ ls
AUTHORS bowtie2-align-l-debug bowtie2-inspect
LICENSE bowtie2-align-s bowtie2-inspect-l
MANUAL bowtie2-align-s-debug bowtie2-inspect-l-debug
MANUAL.markdown bowtie2-build bowtie2-inspect-s
NEWS bowtie2-build-l bowtie2-inspect-s-debug
TUTORIAL bowtie2-build-l-debug doc
VERSION bowtie2-build-s example
bowtie2 bowtie2-build-s-debug scripts
bowtie2-align-l bowtie2-buildc
Mds-MacBook-Pro:bowtie2-2.2.3 ZILLURRAHMAN$ cd example
Mds-MacBook-Pro:example ZILLURRAHMAN$ ls
commands index reads reference
Mds-MacBook-Pro:example ZILLURRAHMAN$ cd index
Mds-MacBook-Pro:index ZILLURRAHMAN$ ls
lambda_virus.1.bt2 lambda_virus.4.bt2 lambdaout
lambda_virus.2.bt2 lambda_virus.rev.1.bt2
lambda_virus.3.bt2 lambda_virus.rev.2.bt2
Mds-MacBook-Pro:index ZILLURRAHMAN$ cd ..
Mds-MacBook-Pro:example ZILLURRAHMAN$ cd reads
Mds-MacBook-Pro:reads ZILLURRAHMAN$ ls
longreads.fq reads_1.fq reads_2.fq simulate.pl
Mds-MacBook-Pro:reads ZILLURRAHMAN$ cd ..
Mds-MacBook-Pro:example ZILLURRAHMAN$ cd reference/
Mds-MacBook-Pro:reference ZILLURRAHMAN$ ls
lambda_virus.fa lambdaout.4.bt2 out
lambdaout.1.bt2 lambdaout.rev.1.bt2 out.sam
lambdaout.2.bt2 lambdaout.rev.2.bt2
lambdaout.3.bt2 nohup.out
Mds-MacBook-Pro:reference ZILLURRAHMAN$ cd ..
Mds-MacBook-Pro:example ZILLURRAHMAN$ open commands
Mds-MacBook-Pro:example ZILLURRAHMAN$ nohup bowtie2 -x lambdaout -q ../reads/reads_1.fq, ../reads/reads_2.fq -S out.sam
appending output to nohup.out
Mds-MacBook-Pro:example ZILLURRAHMAN$ tail nohup.out
Extra parameter(s) specified: "../reads/reads_2.fq"
Error: Encountered internal Bowtie 2 exception (#1)
Command: /Users/ZILLURRAHMAN/Desktop/Bioinformatics/Bow-tie/bowtie2-2.2.3/bowtie2-align-s --wrapper basic-0 -x lambdaout -q -S out.sam ../reads/reads_1.fq, ../reads/reads_2.fq
(ERR): bowtie2-align exited with value 1
Mds-MacBook-Pro:example ZILLURRAHMAN$ ls
commands index nohup.out reads reference
Mds-MacBook-Pro:example ZILLURRAHMAN$

[GenoMax]

In this case the index is already pre-built for lambda virus (the files in the index directory).

I would suggest getting prebuilt sequence/index/annotation bundle from Illumina iGenomes site if you are working with a genome available there: http://support.illumina.com/sequenci...e/igenome.html. Otherwise you can follow the directions here to build a bowtie2 index: http://bowtie-bio.sourceforge.net/bo...-build-indexer