bowtie2 results with NGS data

Question

[adnan.sabar7865]

Hello,

I made index from my reference file and run command to align my metagenomic data by bowtie2. command is

bowtie2 -x <index_referance> -1 <paired_end_read_path_1.fastq> -2 <paired_end_read_path_2.fastq> -s <outputname.sam> and i got this result on screen without getting sam file in output folder.

16190304 reads; of these:
  16190304 (100.00%) were paired; of these:
    16189692 (100.00%) aligned concordantly 0 times
    612 (0.00%) aligned concordantly exactly 1 time
    0 (0.00%) aligned concordantly >1 times
    ----
    16189692 pairs aligned concordantly 0 times; of these:
      59 (0.00%) aligned discordantly 1 time
    ----
    16189633 pairs aligned 0 times concordantly or discordantly; of these:
      32379266 mates make up the pairs; of these:
        32379200 (100.00%) aligned 0 times
        66 (0.00%) aligned exactly 1 time
        0 (0.00%) aligned >1 times
0.00% overall alignment rate

Can anybody tell me what are these results and what's wrong with this?? please

[Chris Dean]

The reason why this might not be producing the expected SAM file could be because the -s option (lowercase) should be -S (uppercase). You can quickly browse the Bowtie2 manual to learn more about these parameters.

Depending on what you are doing, an overall alignment rate of 0.00% may not be a bad thing. For example, if you want to identify and remove host-associated reads from your metagenomic data, then this alignment rate is what you want -- little evidence of host DNA contamination.

You may also consider using Kraken2. The speed is much faster for metagenomic data.

[lieven.sterck]

well spotted, though would that not only affect the output-writing part? (and would thus the numbers not be ok even if the output could not be generated?)

[Chris Dean]

Not sure. I am surprised the program did not raise an exception if in fact the -s option was used with a string and not an int. Should definitely rerun the command with the suggested parameters and see what happens. Some clarification on why bowtie2 is being used over commonly used metagenomic classifiers would also be helpful.

[lieven.sterck]

good point!

ah, and the fact it being metagenomic data can indeed turn out to be important and perhaps the cause of the observation. (missed that in my initial reading, my bad)

[lieven.sterck]

it means that nothing was able to align .

that should not be so I suggest you carefully check your files (names) , paths and settings in your commandline

Is the index to align to correct? (no message there when creating it?)

[adnan.sabar7865]

when i run this command it took a lot of time to completion and then got that message that i showed up. but in directory there is no more file and just only the old ones for index. How i can check this?