[maivantan]

Hi all,
I am analyze fastq, i made with reference lambda file first. but it did not work as:

USER-no-MacBook-Pro-4:tan_analysis user$ bowtie2 -x lambda_virus -U /example/reads/reads_1.fq -S eg1.sam
Warning: Could not open read file "/example/reads/reads_1.fq" for reading; skipping...
Error: No input read files were valid
bowtie2-align exited with value 1

Anyone can help me?
Thank you

[dpryan]

As the error message says, "/example/reads/reads_1.fq" doesn't exist. Try specifying a file that exists.

[maivantan]

but all the files of lambda_virus stayed in the same directory tan_analysis.

[dpryan]

but all the files of lambda_virus stayed in the same directory tan_analysis.

What's your point? If the fastq files are there too, then specify that and not some other directory. If the fastq files are elsewhere, then specify that correctly. It's finding the indices you created just fine, just not the fastq file.

[maivantan]

Here is my files for lambda_virus

SER-no-MacBook-Pro-4:tan_analysis user$ cd bowtie2-2.1.0/
USER-no-MacBook-Pro-4:bowtie2-2.1.0 user$ ls
AUTHORS bowtie2 doc
LICENSE bowtie2-align eg1.sam
MANUAL bowtie2-align-debug eg2.sam
MANUAL.markdown bowtie2-build example
NEWS bowtie2-build-debug scripts
TUTORIAL bowtie2-inspect
VERSION bowtie2-inspect-debug
USER-no-MacBook-Pro-4:bowtie2-2.1.0 user$ cd example/
USER-no-MacBook-Pro-4:example user$ ls
index reads reference
USER-no-MacBook-Pro-4:example user$


Please help me
Thank you

[dpryan]

If you're in the bowtie2-2.1.0 directory:

./bowtie2 -x example/index/lambda_virus -U example/reads/reads_1.fq -S eg1.sam

[maivantan]

I sorry to disturb you but i got this problem

USER-no-MacBook-Pro-4:bowtie2-2.1.0 user$ ./bowtie2 -x example/index/lamda_virus -U example/reads/reads_1.fq -S eg1.sam
Could not locate a Bowtie index corresponding to basename "example/index/lamda_virus"
Error: Encountered internal Bowtie 2 exception (#1)
Command: /Users/user/tan_analysis/bowtie2-2.1.0/bowtie2-align --wrapper basic-0 -x example/index/lamda_virus -S eg1.sam -U example/reads/reads_1.fq
bowtie2-align exited with value 1

please help me.
Thank you

[dpryan]

There was a "b" missing in my command, it should be:

./bowtie2 -x example/index/lambda_virus -U example/reads/reads_1.fq -S eg1.sam

Edit: Actually, it was just missing from yours, not mine.

[maivantan]

yes, it work now.
Thank you very much

[bioGal]

fastQC shows overduplication of library

Hi All,
I have a different question about fastQC reports.
I used the fastQC kit to determine read quality of my libraries and everything looked good except for the level of duplication, which was very high in all of my libraries (see attached pic).
It seems to me that having such a high level of duplication would affect differential gene expression analysis. My question is, is this something I should be worried about? And if so what can I do to fix the issue.
thanks
Mila

[dpryan]

1) You should start a new thread.

2) That's not unusual for RNAseq, particularly if you did total RNA, where most of the reads originate from (rather short) rRNA.