Aligning multiple fastq files in Bowtie2

Question

[vineethmails]

I am trying to align multiple fastq file in bowtie2. I am using the following command line code

 ./bowtie2 -t -x bss  -1 C52.1.fastq,C55.1.fastq -2 C52.2.fastq,C55.2.fastq -S C5255.sam

But its seems that only the first file is getting aligned. the out put is showing results like this

Time loading reference: 00:00:07
Time loading forward index: 00:00:18
Time loading mirror index: 00:00:11
Multiseed full-index search: 00:00:38
16906 reads; of these:
  16906 (100.00%) were paired; of these:
    5902 (34.91%) aligned concordantly 0 times
    8736 (51.67%) aligned concordantly exactly 1 time
    2268 (13.42%) aligned concordantly >1 times
    ----
    5902 pairs aligned concordantly 0 times; of these:
      1170 (19.82%) aligned discordantly 1 time
    ----
    4732 pairs aligned 0 times concordantly or discordantly; of these:
      9464 mates make up the pairs; of these:
        5117 (54.07%) aligned 0 times
        2813 (29.72%) aligned exactly 1 time
        1534 (16.21%) aligned >1 times
84.87% overall alignment rate
Time searching: 00:01:14
Overall time: 00:01:15

When i run it using only one pair of read, the output is similar

$ ./bowtie2 -t -x bss -1 C52.1.fastq -2 C52.2.fastq -S C52.sam
Time loading reference: 00:00:08
Time loading forward index: 00:00:18
Time loading mirror index: 00:00:14
Multiseed full-index search: 00:00:38
16906 reads; of these:
  16906 (100.00%) were paired; of these:
    5902 (34.91%) aligned concordantly 0 times
    8736 (51.67%) aligned concordantly exactly 1 time
    2268 (13.42%) aligned concordantly >1 times
    ----
    5902 pairs aligned concordantly 0 times; of these:
      1170 (19.82%) aligned discordantly 1 time
    ----
    4732 pairs aligned 0 times concordantly or discordantly; of these:
      9464 mates make up the pairs; of these:
        5117 (54.07%) aligned 0 times
        2813 (29.72%) aligned exactly 1 time
        1534 (16.21%) aligned >1 times
84.87% overall alignment rate
Time searching: 00:01:18

Why is it so. I am new to bioinformatics. Any help is appreciated.

Thanks

[Carlo Yague]

What happens if you call bowtie on the other set of fastq files ?

./bowtie2 -t -x bss -1 C55.1.fastq -2 C55.2.fastq -S C55.sam

[vineethmails]

$ ./bowtie2 -t -x bss -1 C55.1.fastq  -2 C55.2.fastq -S C55.sam
Time loading reference: 00:00:07
Time loading forward index: 00:00:18
Time loading mirror index: 00:00:13
Multiseed full-index search: 00:01:10
28255 reads; of these:
  28255 (100.00%) were paired; of these:
    7675 (27.16%) aligned concordantly 0 times
    15831 (56.03%) aligned concordantly exactly 1 time
    4749 (16.81%) aligned concordantly >1 times
    ----
    7675 pairs aligned concordantly 0 times; of these:
      1384 (18.03%) aligned discordantly 1 time
    ----
    6291 pairs aligned 0 times concordantly or discordantly; of these:
      12582 mates make up the pairs; of these:
        7543 (59.95%) aligned 0 times
        3169 (25.19%) aligned exactly 1 time
        1870 (14.86%) aligned >1 times
86.65% overall alignment rate
Time searching: 00:01:48
Overall time: 00:01:48

It gives this result

[Satyajeet Khare]

Whats your bowtie2 version? Can you try updating it? If the problem persists (this is not an answer to your question) you can concatenate the fastq files and run Bowtie2.

[vineethmails]

 $ ./bowtie2 -version
Bowtie 2 version 2.3.1 by Ben Langmead ([e-mail], www.cs.jhu.edu/~langmea)

[vineethmails]

i tried the commands on the latest version 2.3.3 , still showing the same results.