bowtie2

Question

[bestone]

Hello, I wrote this code

(test) ➜  dene bowtie2 --very-fast-local -x /Users/uguremre/Desktop/dene/bowtie2_index/bowtie2 -1 /Users/uguremre/Desktop/dene/B11_L03_57_1.fq.gz -2 /Users/uguremre/Desktop/dene/B11_L03_57_2.fq.gz -S /Users/uguremre/Desktop/dene/B11.sam

But I got this ERROR. How can I figure out this issue? Could you pls help me?

(ERR): "/Users/uguremre/Desktop/dene/bowtie2_index/bowtie2" does not exist or is not a Bowtie 2 index
Exiting now ...
(test) ➜  dene ls

[GenoMax]

It would be not so logical to use bowtie2 as basename of your index since that also happens to be a program name. When you create the index use some other name as the basename if that is what you did above.

Otherwise show us a listing from ls -lhR /Users/uguremre/Desktop/dene/bowtie2_index/*.

[bestone]

It says

zsh: no matches found: /Users/uguremre/Desktop/dene/bowtie2_index/*

[GenoMax]

Looks like that is not the location of bowtie2 indexes. Please use the correct path in your command line above. Only provide the basename of the index.

[bestone]

I created a new folder on my desktop whose name is dene. In this folder, I have my reference data Prunus_armeniaca_cv_Stella.fasta.gz and my other fastq data which is B11_L03_57_1.fq.gz and B11_L03_57_2.fq.gz and to create bowtie2_ index I used this code

bowtie2-build *  Prunus_armeniaca_cv_Stella.fasta.gz bt2_index_base

It created bt2_index_base.3.bt2l.tmp and bowtie.2.bt2.tmp this type four or five files and I tried to run bowtie2. I used this code

bowtie2 --very-fast-local -x /Users/uguremre/Desktop/dene/bowtie2_index/bowtie2 -1 /Users/uguremre/Desktop/dene/B11_L03_57_1.fq.gz -2 /Users/uguremre/Desktop/dene/B11_L03_57_2.fq.gz -S /Users/uguremre/Desktop/dene/B11.sam

but I got ERROR. How can I fix it?

[GenoMax]

That is rather unfortunate choice of name but you called your index with basename bt2_index_base. So in your code you will need to use -x /Users/uguremre/Desktop/dene/bowtie2_index/bt2_index_base. This will work as long as your index was created without any errors.

[bestone]

(ERR): "/Users/uguremre/Desktop/dene/bowtie2_index/bt2_index_base" does not exist or is not a Bowtie 2 index. Unfortunately, I have got the same ERROR:(

Answer 1

[ATpoint]

It created bt2_index_base.3.bt2l.tmp and bowtie.2.bt2.tmp

The tmp suffix indicates that the process has not finished properly. It might have been killed due to memory problems. Please show the last few lines of the indexing log file, so what bowtie2 prints to screen during indexing.

[bestone]

I used this code " ➜ dene bowtie2 -x /Users/uguremre/Desktop/dene/bowtie2_index/bt2_index_base -1 /Users/uguremre/Desktop/dene/B11_L03_57_1.fq.gz -2 /Users/uguremre/Desktop/dene/B11_L03_57_2.fq.gz -S /Users/uguremre/Desktop/dene/B11.sam it has given this error. there is no more information from lines (ERR): "/Users/uguremre/Desktop/dene/bowtie2_index/bt2_index_base" does not exist or is not a Bowtie 2 index Exiting now ...

[ATpoint]

Please read my answer. The indexing did not finish properly. Please show what the indexing process printed, not the alignment.

[bestone]

"bt2_index_base.3.bt2l.tmp , bt2_index_base.4.bt2l.tmp, bt2_index_base.1.bt2l.tmp, bt2_index_base.2.bt2l.tmp, bowtie.2.bt2.tmp, bowtie.3.bt2.tmp, bowtie.4.bt2.tmp". It has these folders.

[GenoMax]

There should be no .tmp files left when the indexing completes successfully. It is possible that you don't have enough RAM to complete this process. What genome is this and what is the expected size? How much RAM do you have available?

[bestone]

I am using a MacBook pro it has 8 GB RAM but maybe you are right laptop is not able to have enough memory. It is the apricot genome. Normally, I have 32 genomes in apricot but I was using just one genome for practice (to learn) which is almost 5 GB "B11_L03_57_1.fq.gz". I run this code "bowtie2-build * Prunus_armeniaca_cv_Stella.fasta.gz bowtie2" I got this

Settings: Output files: "bowtie2..bt2l" Line rate: 7 (line is 128 bytes) Lines per side: 1 (side is 128 bytes) Offset rate: 4 (one in 16) FTable chars: 10 Strings: unpacked Max bucket size: default Max bucket size, sqrt multiplier: default Max bucket size, len divisor: 4 Difference-cover sample period: 1024 Endianness: little Actual local endianness: little Sanity checking: disabled Assertions: disabled Random seed: 0 Sizeofs: void:8, int:4, long:8, size_t:8 Input files DNA, FASTA: B11_L03_57_1.fq.gz B11_L03_57_2.fq.gz Prunus_armeniaca_cv_Stella.fasta.gz Prunus_armeniaca_cv_Stella.fasta.gz Building a LARGE index Reading reference sizes Warning: Encountered empty reference sequence Warning: Encountered empty reference sequence Time reading reference sizes: 00:04:07 Calculating joined length Writing header Reserving space for joined string Joining reference sequences Reference file does not seem to be a FASTA file Time to join reference sequences: 00:00:24 Total time for call to driver() for forward index: 00:04:56 Error: Encountered internal Bowtie 2 exception (#1) Command: /Users/uguremre/opt/anaconda3/envs/test/bin/bowtie2-build-l --wrapper basic-0 B11_L03_57_1.fq.gz,B11_L03_57_2.fq.gz,Prunus_armeniaca_cv_Stella.fasta.gz,Prunus_armeniaca_cv_Stella.fasta.gz bowtie2 Deleting "bowtie2.3.bt2l" file written during aborted indexing attempt. Deleting "bowtie2.4.bt2l" file written during aborted indexing attempt. Deleting "bowtie2.1.bt2l" file written during aborted indexing attempt. Deleting "bowtie2.2.bt2l" file written during aborted indexing attempt.

[ATpoint]

Some basics:

You index a reference genome in fastA format. That produces the index to align your fastQ files against. You do not index fastQ files. Please also learn to read logs and error messages, what you pasted clearly tells you that something went wrong. Rerun the indexing with only that fasta file. Then do the alignment again.

bowtie2-build Prunus_armeniaca_cv_Stella.fasta.gz idx

That will (if 8GB is enough for this genome, it might not) produce index files with basename idx.

[bestone]

Thank you for replying guys. But it has still given the same error.

[ATpoint]

If you applied any of what was said the error during indexing would change. I am out here since this does not lead anywhere. Maybe someone locally can help hands-on.

[bestone]

I tried this code as you send before. "bowtie2-build Prunus_armeniaca_cv_Stella.fasta.gz idx" and I got six folders like these "idx.3.bt2" ,"idx.4.bt2", " idx.1.bt2", " idx.2.bt2", " idx.rev.1.bt2", "idx.rev.2.bt2"

and then I run this code;

bowtie2 -x /Users/uguremre/Desktop/dene/bowtie2_index/idx -1 /Users/uguremre/Desktop/dene/B11_L03_57_1.fq.gz -2 /Users/uguremre/Desktop/dene/B11_L03_57_2.fq.gz -S /Users/uguremre/Desktop/dene/B11.sam

It gives error; (ERR): "/Users/uguremre/Desktop/dene/bowtie2_index/idx" does not exist or is not a Bowtie 2 index Exiting now ...

[GenoMax]

Show us a listing of

ls -lh /Users/uguremre/Desktop/dene/bowtie2_index/idx*

[bestone]

It says : zsh: no matches found: /Users/uguremre/Desktop/dene/bowtie2_index/idx*

[GenoMax]

Then we are back to square one. You have either not created the indexes and/or they are not located in the directory where you think they are. If you are new to using the command line/terminal then it would be best to spend some time learning UNIX basics. This is a useful tutorial: http://korflab.ucdavis.edu/unix_and_perl/current.html#part1

[bestone]

all data are here under the dene folder's name.

[GenoMax]

Your index files are not in a directory called bowtie2_index. They are now directly in dene directory.

Try this command

bowtie2 -x /Users/uguremre/Desktop/dene/idx -1 /Users/uguremre/Desktop/dene/B11_L03_57_1.fq.gz -2 /Users/uguremre/Desktop/dene/B11_L03_57_2.fq.gz -S /Users/uguremre/Desktop/dene/B11.sam

[bestone]

Perfect. I think that it worked. If I get any issues I will let you know. Btw, thank you so much for replying and helping.