GBS:stakcs software error in ref_map.pl

[meghavarshney]

Hello Everyone,

I am currently analysing GBS data using stacks software as i ma currently facing some problem with ref_map.pl.
According to this script not getting proper output for this run.

ref_map.pl version 1.37 started at 2016-04-29 16:52:29
/usr/local/bin/ref_map.pl -T 4 -m 3 -n 1 -b 2 -o ref_out/ -S -s RG-1.bam
Identifying unique stacks; file   1 of   1 [RG-1]
/usr/local/bin/pstacks -t bam -f RG-1.bam -o ref_out -i 1 -p 4 -m 3 2>&1
Min depth of coverage to report a stack: 3
Model type: SNP
Alpha significance level for model: 0.05
BAM support was not enabled when Stacks was compiled.
Parsing RG-1.bam
  Analyzed 0 sequence reads; Identified 0 unique stacks from those reads.
  Merged 0 unique Stacks into 0 loci.
Identifying polymorphic sites and calling consensus sequences...done.
  Number of utilized reads 0
  Mean coverage depth is -nan; Std Dev: -0; Max: 0
Writing loci, SNPs, alleles to 'ref_out/...'
  Wrote 0 loci, excluded 0 loci due to insuffient depth of coverage; blacklisted 0 loci.
/usr/local/bin/cstacks -g -b 2 -o ref_out -s ref_out/RG-1  -p 4 -n 1 2>&1
Number of mismatches allowed between stacks: 1
Loci matched based on genomic location.
Constructing catalog from 1 samples.
Initializing new catalog...
  Parsing ref_out/RG-1.tags.tsv.gz
  Parsing ref_out/RG-1.snps.tsv.gz
  Parsing ref_out/RG-1.alleles.tsv.gz
Building an index of the catalog.
Writing catalog to 'ref_out/... done.
/usr/local/bin/sstacks -g -b 2 -c ref_out/batch_2 -o ref_out -s ref_out/RG-1  -p 4 2>&1
Searching for matches by genomic location...
  Parsing ref_out/batch_2.catalog.tags.tsv.gz
  Parsing ref_out/batch_2.catalog.snps.tsv.gz
  Parsing ref_out/batch_2.catalog.alleles.tsv.gz
Processing sample 'ref_out/RG-1' [1 of 1]
  Parsing ref_out/RG-1.tags.tsv.gz
  Parsing ref_out/RG-1.snps.tsv.gz
  Parsing ref_out/RG-1.alleles.tsv.gz
/usr/local/bin/populations -b 2 -P ref_out -s -t 4 2>&1
A population map was not specified, all samples will be read from 'ref_out/' as a single popultaion.
Fst kernel smoothing: off
Bootstrap resampling: off
Percent samples limit per population: 0
Locus Population limit: 1
Minimum stack depth: 0
Log liklihood filtering: off; threshold: 0
Minor allele frequency cutoff: 0
Maximum observed heterozygosity cutoff: 1
Applying Fst correction: none.
No population map specified, building file list.
Found 2 input file(s).
  1 population found
    1: RG-1, RG-1.sorted
  1 group of populations found
    1: 1
  Parsing ref_out/batch_2.catalog.tags.tsv.gz
  Parsing ref_out/batch_2.catalog.snps.tsv.gz
  Parsing ref_out/batch_2.catalog.alleles.tsv.gz
Catalog is not reference aligned, arbitrarily ordering catalog loci.
 Unable to open 'ref_out/RG-1'
Warning: unable to find any matches in file 'RG-1', excluding this sample from population analysis.
 Unable to open 'ref_out/RG-1.sorted'
Warning: unable to find any matches in file 'RG-1.sorted', excluding this sample from population analysis.
Populating observed haplotypes for 0 samples, 0 loci.
Writing SQL markers file to 'ref_out/batch_2.markers.tsv'
Removing 0 loci that did not pass sample/population constraints... retained 0 loci.
refmap_map.pl completed at 2016-04-29 16:52:29

this is the log file information form ref_map.pl

[SNPsaurus]

I think this error is the key "BAM support was not enabled when Stacks was compiled.
Parsing RG-1.bam
Analyzed 0 sequence reads; Identified 0 unique stacks from those reads."

You supplied a BAM file, but when Stacks was compiled it didn't include components for reading BAM files. I'd look at the install instructions for how to include BAM support.

[meghavarshney]

as after your suggestion i tried to unable bam file .
as i was not able to do this in a proper way, so i just tried to reinstall it
with these commands

[root@bioinfo3-c stacks-1.40]# ./configure --enable-bam --with-bam-include-path=/usr/local/include/bam/ --with-bam-lib-path=/usr/local/lib/ --enable-sparsehash --with-sparsehash-include-path=/usr/local/include/sparsehash/
checking for a BSD-compatible install... /usr/bin/install -c
checking whether build environment is sane... yes
checking for a thread-safe mkdir -p... /usr/bin/mkdir -p
checking for gawk... gawk
checking whether make sets $(MAKE)... yes
checking whether make supports nested variables... yes
checking for g++... g++
checking whether the C++ compiler works... yes
checking for C++ compiler default output file name... a.out
checking for suffix of executables... 
checking whether we are cross compiling... no
checking for suffix of object files... o
checking whether we are using the GNU C++ compiler... yes
checking whether g++ accepts -g... yes
checking for style of include used by make... GNU
checking dependency style of g++... gcc3
checking for gcc... gcc
checking whether we are using the GNU C compiler... yes
checking whether gcc accepts -g... yes
checking for gcc option to accept ISO C89... none needed
checking whether gcc understands -c and -o together... yes
checking dependency style of gcc... gcc3
checking whether g++ supports C++11 features by default... no
checking whether g++ supports C++11 features with -std=gnu++11... yes
checking for omp_set_num_threads in -lgomp... yes
checking for gzread in -lz... yes
checking for dirent.h that defines DIR... yes
checking for library containing opendir... none required
checking how to run the C preprocessor... gcc -E
checking for grep that handles long lines and -e... /usr/bin/grep
checking for egrep... /usr/bin/grep -E
checking for ANSI C header files... yes
checking for sys/types.h... yes
checking for sys/stat.h... yes
checking for stdlib.h... yes
checking for string.h... yes
checking for memory.h... yes
checking for strings.h... yes
checking for inttypes.h... yes
checking for stdint.h... yes
checking for unistd.h... yes
checking float.h usability... yes
checking float.h presence... yes
checking for float.h... yes
checking limits.h usability... yes
checking limits.h presence... yes
checking for limits.h... yes
checking for stdlib.h... (cached) yes
checking for string.h... (cached) yes
checking for gcc option to support OpenMP... -fopenmp
checking for stdbool.h that conforms to C99... yes
checking for _Bool... yes
checking for an ANSI C-conforming const... yes
checking for size_t... yes
checking whether closedir returns void... no
checking for stdlib.h... (cached) yes
checking for GNU libc compatible malloc... yes
checking for stdlib.h... (cached) yes
checking for GNU libc compatible realloc... yes
checking for floor... no
checking for memset... yes
checking for pow... no
checking for sqrt... no
checking for gawk... (cached) gawk
checking that generated files are newer than configure... done
configure: creating ./config.status
config.status: creating Makefile
config.status: creating config.h
config.status: config.h is unchanged
config.status: executing depfiles commands
[root@bioinfo3-c stacks-1.40]# 
make  all-am
make[1]: Entering directory `/mnt/bioinfo3/Desktop/Megha/tools/new_stacks/stacks-1.40'
g++ -fopenmp -I/usr/local/include/bam/ -I/usr/local/include/sparsehash/ -g -O2 -std=gnu++11 -fopenmp  -o pstacks src/pstacks-pstacks.o src/pstacks-stacks.o src/pstacks-mstack.o src/pstacks-DNANSeq.o src/pstacks-DNASeq.o src/pstacks-models.o src/pstacks-utils.o src/pstacks-input.o /usr/local/lib//libbam.a -lz -lgomp 
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(bgzf.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(bam.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(bam_import.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(knetfile.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(sam_header.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(kstring.o)' is incompatible with i386:x86-64 output
/usr/bin/ld: i386 architecture of input file `/usr/local/lib//libbam.a(bam_aux.o)' is incompatible with i386:x86-64 output
collect2: error: ld returned 1 exit status
make[1]: *** [pstacks] Error 1
make[1]: Leaving directory `/mnt/bioinfo3/Desktop/Megha/tools/new_stacks/stacks-1.40'
make: *** [all] Error 2
[root@bioinfo3-c stacks-1.40]# 

in make i am getting these error . could you please explain it ..