BWA - Error in Run Time

[Arupsss]

I have installed BWA. And build index of hg19 using command:

bwa index -a bwtsw hg19.fa

Now I am trying to find alignment using command:

$ ./bwa aln hg.fa SRR493081_1.fastq > aln_sa.sai
[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln] fail to locate the index
[main] Version: 0.6.2-r126
[main] CMD: ./bwa aln hg.fa SRR493081_1.fastq
[main] Real time: 0.000 sec; CPU: 0.010 sec

However, gives above error. Can anybody helps me why is this giving such error.

[Applemelon]

According to your statement,
this commond
$ ./bwa aln hg.fa SRR493081_1.fastq > aln_sa.sai

should be

$ ./bwa aln hg19.fa SRR493081_1.fastq > aln_sa.sai

Because hg.fa is different with hg19.fa, bwa can't find the index.

[kellywilliams]

I have a similar problem and cannot get bwa alignment to work.

I completed the index successfully

$ bwa index -a bwtsw -p hg19 hg19.fa
bwa_index] Pack FASTA... 27.96 sec
[bwa_index] Construct BWT for the packed sequence...
[BWTIncCreate] textLength=6274322528, availableWord=453484340
[BWTIncConstructFromPacked] 10 iterations done. 100000000 characters processed.
[BWTIncConstructFromPacked] 20 iterations done. 200000000 characters processed.
.
.
.
[BWTIncConstructFromPacked] 690 iterations done. 6264217232 characters processed.
[bwt_gen] Finished constructing BWT in 695 iterations.
[bwa_index] 3556.44 seconds elapse.
[bwa_index] Update BWT... 21.30 sec
[bwa_index] Pack forward-only FASTA... 18.66 sec
[bwa_index] Construct SA from BWT and Occ... 1118.66 sec
[main] Version: 0.7.5a-r405
[main] CMD: bwa index -a bwtsw -p hg19 hg19.fa
[main] Real time: 4768.228 sec; CPU: 4743.022 sec

then when I attempted the alignment:

$ bwa aln -q 28 -t 12 hg19.fa sample1_1.fastq.gz > sample1_1.sai
[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln] fail to locate the index

Even when I use the absolute path i get :

$ bwa aln -q 28 -t 12 /NGSsoftware/hg19.fa 8-A182_1.fastq.gz > 8-A182_1.sai
[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln] fail to locate the index

I was wondering if there were permission problems but even after changing permissions of to all users rwx it still will not work.

Any suggestions? I am using Mac OSX 10.8.4 with Xcode installed.

[GenoMax]

Since you used "-p hg19" as the prefix for the output database you need to include that as the prefix for the database.

Try the following:

$ bwa aln -q 28 -t 12 /NGSsoftware/hg19 8-A182_1.fastq.gz > 8-A182_1.sai

[kellywilliams]

Since you used "-p hg19" as the prefix for the output database you need to include that as the prefix for the database.

Try the following:

$ bwa aln -q 28 -t 12 /NGSsoftware/hg19 8-A182_1.fastq.gz > 8-A182_1.sai

Thank you GenoMax, this worked perfectly until I tried to perform bwa sampe:

$ bwa sampe –r '@RG\tID:sample8\tLB:sample8\tSM:sample8\tPL:ILLUMINA' /NGSsoftware/hg19  8-A182_1.sai  8-A182_2.sai 8-A182_1.fastq.gz 8-A182_2.fastq.gz 8-A182.sam
[bwa_sai2sam_pe] fail to locate the index

If I remove the -r argument, bwa sampe will run, but I wish to have the read groups added.

[GenoMax]

Can you post a listing of the files in the /NGSsoftware directory?

$ ls -l /NGSsoftware/hg19*

[kellywilliams]

Here are the files

$ ls -l /NGSsoftware/hg19*
-rw-r--r--  1 kwilliams  wheel        6693  5 Sep 13:38 /NGSsoftware/hg19.amb
-rw-r--r--  1 kwilliams  wheel        3635  5 Sep 13:38 /NGSsoftware/hg19.ann
-rw-r--r--  1 kwilliams  wheel  3101804844  5 Sep 13:38 /NGSsoftware/hg19.bwt
-rw-r--r--  1 kwilliams  wheel  3163842112  5 Sep 08:06 /NGSsoftware/hg19.fa
-rw-r--r--  1 kwilliams  wheel   775451187  5 Sep 13:38 /NGSsoftware/hg19.pac
-rw-r--r--  1 kwilliams  wheel  1550902424  5 Sep 13:57 /NGSsoftware/hg19.sa

[kellywilliams]

Hi, just wondering if anyone has insight into the problem above regarding read groups and bwa?
Many thanks.

[GenoMax]

Have you tried to run the command with double quotes (") instead of single?

$ bwa sampe –r "@RG\tID:sample8\tLB:sample8\tSM:sample8\tPL:ILLUMINA" /NGSsoftware/hg19  8-A182_1.sai  8-A182_2.sai 8-A182_1.fastq.gz 8-A182_2.fastq.gz 8-A182.sam

It is confusing that you chose to use "hg19" as your prefix name for the indexes.

[kellywilliams]

Yes, that also comes up with

[bwa_sai2sam_pe] fail to locate the index

[GenoMax]

Should have asked this before. Are you using a Mac? Did you compile bwa yourself?

[kellywilliams]

Yes I am using a mac. I have Xcode installed and compiled bwa using Homebrew.

[GenoMax]

There were some problems with bwa on a Mac in past (http://seqanswers.com/forums/showthread.php?t=24133). I am not sure if those persist/are still relevant. Perhaps someone else who is currently running the latest bwa (0.7.5) on Mac can confirm.

What version of OS X (and Xcode) are you running?