Bowtie2: Mate file error & internal exception

Question

[stefan]

Hello, I am a bachelor student (biomedical sciences). We are doing a Illumina sequencing task to find out a subjects heritage. Currently, I am struggling with our alignment step and I can't manage to find the answer anywhere. This is my code:

bowtie2 --threads 10  -x Reference/Bowtie2/homosapiens  -1 trimOutput/lc_good_1.fastq  -2 trimOutput/lc_good_2.fastq -S  > alignmentOutput/lc.bowtie.sam

And the error messages:

 Error: 0 mate files/sequences were specified with -1, but 1
mate files/sequences were specified with -2.  The same number of mate files/
sequences must be specified with -1 and -2.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /cm/shared/apps/bowtie2/2.4.2/bin/../src/bowtie2-2.4.2/bowtie2-align-s --wrapper basic-0 --threads 10 -x Reference/Bowtie2/homosapiens -S -1 -2 trimOutput/lc_good_2.fastq trimOutput/lc_good_1.fastq
(ERR): bowtie2-align exited with value 1

I have read the bowtie2 -help page a hunder times but can't seem to find the solution. Any help is much appreciated!

This is the first time I'm working with a programming language so use simple words in your response :)

Answer 1

[GenoMax]

Can you try

bowtie2 --threads 10  -x Reference/Bowtie2/homosapiens  -1 trimOutput/lc_good_1.fastq  -2 trimOutput/lc_good_2.fastq -S  alignmentOutput/lc.bowtie.sam

There is no need to use a > before a file name.

You can actually pipe the output directly to samtools and omit making SAM format file altogether. This saves space.

bowtie2 --threads 10  -x Reference/Bowtie2/homosapiens  -1 trimOutput/lc_good_1.fastq  -2 trimOutput/lc_good_2.fastq  | samtools sort -o alignment.bam -

[stefan]

I tried

bowtie2 --threads 10  -x Reference/Bowtie2/homosapiens  -1 trimOutput/lc_good_1.fastq  -2 trimOutput/lc_good_2.fastq -S  alignmentOutput/lc.bowtie.sam

and still got

Error reading _ebwt[] array: no more data
Error: Encountered internal Bowtie 2 exception (#1)
Command: /cm/shared/apps/bowtie2/2.4.2/bin/../src/bowtie2-2.4.2/bowtie2-align-s --wrapper basic-0 --threads 10 -x Reference/Bowtie2/homosapiens -S alignmentOutput/lc.bowtie.sam -1 trimOutput/lc_good_1.fastq -2 trimOutput/lc_good_2.fastq
(ERR): bowtie2-align exited with value 1

Tried

bowtie2 --threads 10  -x Reference/Bowtie2/homosapiens  -1 trimOutput/lc_good_1.fastq  -2 trimOutput/lc_good_2.fastq  | samtools sort -o alignment.bam -

And got

Error reading _ebwt[] array: no more data
Error: Encountered internal Bowtie 2 exception (#1)
Command: /cm/shared/apps/bowtie2/2.4.2/bin/../src/bowtie2-2.4.2/bowtie2-align-s --wrapper basic-0 --threads 10 -x Reference/Bowtie2/homosapiens -1 trimOutput/lc_good_1.fastq -2 trimOutput/lc_good_2.fastq
(ERR): bowtie2-align exited with value 1
samtools sort: failed to read header from "-"

Also tried without the "-" at the end of the command, but didn't change anything.

[quentin54520]

For your reference genome -x Reference/Bowtie2/homosapiens homosapiens is a directory or the basename of your index file? In the directory Reference/Bowtie2 you have files name homosapiens.1.bt2 homosapiens.2.bt2 etc?

[stefan]

It is the basename, see the answer to GenoMax below

[stefan]

It is the basename, see the answer to GenoMax below

[GenoMax]

Can you post the output of ls -l Reference/Bowtie2/homosapiens? As @quentin54520 says you are likely not using the basename of your bowtie2 index files.

[stefan]

    [student@beowulf ~]$ ls Reference/Bowtie2/
homosapiens.1.bt2  homosapiens.2.bt2  homosapiens.3.bt2  homosapiens.4.bt2  homosapiens.rev.1.bt2  homosapiens.rev.2.bt2

As you can see it is a basename of my index file. The bowtie2 manual said I should remove the trailing (.X.bt2) which I think I did by referencing the index with Reference/Bowtie2/homosapiens

[student@beowulf ~]$ ls -l Reference/Bowtie2/homosapiens
ls: cannot access Reference/Bowtie2/homosapiens: No such file or directory

the ls -l gives me a an error. Probably because using ls -l does not specify any of the 6 files that are in the bowtie2 directory.

[GenoMax]

Did you create this index yourself? Generally one needs to have at least a softlink (if not the full file) for the genome multi-fasta file in the directory where the index files are present. Your genome file must have been called homosapiens.fa? Make sure there is a softlink or a copy in index directory and try again.

BTW: ls -l Reference/Bowtie2/homosapiens* should work. I forgot the wildcard before.

[stefan]

I did not create the index myself, my teacher did. I'll ask him whether this might be the problem. As for now, I switched to bowtie2 version 2.2.6. which seems to work. Unfortunately, adding CPUs with --thread or -p does not work in this version for me. I have decided to switch to using BWA alignment.

[jordi.planells]

Have you trimmed the files independently? Maybe you are facing this error cause you have different amount of reads in R1 and R2

[stefan]

I have not trimmed the files seperately. This is what i used for trimming:

prinseq-lite -out_good trimOutput/lc_good -out_bad trim_output/lc_bad -fastq fastq/WGS/WGS7_S1_L001_R1_001.fastq -fastq2 fastq/WGS/WGS7_S1_L001_R2_001.fastq  -trim_right 1 -max_gc 95 -min_len 90 -trim_qual_type mean -trim_qual_window 8 -trim_qual_right 35 -trim_qual_left 35

When doing a fastqc of the trimmed files, i see that both have 506 316 sequences. So there is no difference in reads of R1 and R2