Installation problems Bio-SamTools

[Robby]

Hi everybody,

I would like to install Bio-SamTools 1.41 without root-permissions. I tried the following commands:

perl Build.PL --install_base /home/$User/perl5
./Build

But I received the following error:

Building Bio-SamTools
which: no nmake in ([...])
gcc -g -Wall -O2 -fPIC -o bam2bedgraph bam2bedgraph.o  -L/path/samtools -lbam -lpthread -lm -lz
bam2bedgraph.o: In function `main':
/path/Bio-SamTools-1.41/c_bin/bam2bedgraph.c:62: undefined reference to `hts_idx_load'
bam2bedgraph.o: In function `bam_index_destroy':
/path/samtools/bam.h:449: undefined reference to `hts_idx_destroy'
/path/samtools/libbam.a(bam_aux.o): In function `bam_parse_region':
/path/samtools/bam_aux.c:63: undefined reference to `hts_parse_reg'
/path/samtools/bam_aux.c:67: undefined reference to `bam_name2id'
/path/samtools/libbam.a(bam.o): In function `bam_fetch':
/path/software/samtools/bam.c:84: undefined reference to `bam_init1'
/path/samtools/libbam.a(bam.o): In function `bam_iter_query':
/path/samtools/bam.h:475: undefined reference to `sam_itr_queryi'
/path/samtools/libbam.a(bam.o): In function `bam_iter_read':
/path/samtools/bam.h:476: undefined reference to `hts_itr_next'
/path/samtools/bam.h:476: undefined reference to `bam_read1'
/path/samtools/libbam.a(bam.o): In function `bam_iter_destroy':
/path/samtools/bam.h:477: undefined reference to `hts_itr_destroy'
/path/samtools/libbam.a(bam.o): In function `bam_fetch':
/path/samtools/bam.c:88: undefined reference to `bam_destroy1'
/path/samtools/libbam.a(bam.o): In function `bam_format1':
/path/samtools/bam.c:37: undefined reference to `sam_format1'
/path/samtools/libbam.a(sam.o): In function `samfaipath':
/path/samtools/sam.c:102: undefined reference to `hts_verbose'
/path/samtools/sam.c:103: undefined reference to `fai_build'
/path/samtools/libbam.a(sam.o): In function `sampileup':
/path/samtools/sam.c:77: undefined reference to `bam_init1'
/path/samtools/libbam.a(sam.o): In function `samread':
/path/samtools/sam.h:95: undefined reference to `sam_read1'
/path/samtools/libbam.a(sam.o): In function `sampileup':
/path/samtools/sam.c:88: undefined reference to `bam_destroy1'
/path/samtools/libbam.a(sam.o): In function `samclose':
/path/samtools/sam.c:66: undefined reference to `bam_hdr_destroy'
/path/samtools/sam.c:67: undefined reference to `hts_close'
/path/samtools/libbam.a(sam.o): In function `samopen':
/path/samtools/sam.c:41: undefined reference to `hts_open'
/path/samtools/sam.c:48: undefined reference to `hts_set_fai_filename'
/path/samtools/sam.c:49: undefined reference to `sam_hdr_read'
/path/samtools/sam.c:51: undefined reference to `hts_verbose'
/path/samtools/sam.c:57: undefined reference to `hts_get_format'
/path/samtools/sam.c:57: undefined reference to `sam_hdr_write'
/path/samtools/libbam.a(sam.o): In function `samthreads':
/path/samtools/sam.c:33: undefined reference to `hts_get_format'
/path/samtools/sam.c:34: undefined reference to `bgzf_mt'
/path/samtools/libbam.a(bam_plbuf.o): In function `bam_plbuf_push':
/path/samtools/bam_plbuf.c:61: undefined reference to `bam_plp_push'
/path/samtools/bam_plbuf.c:63: undefined reference to `bam_plp_next'
/path/samtools/libbam.a(bam_plbuf.o): In function `bam_plbuf_destroy':
/path/samtools/bam_plbuf.c:53: undefined reference to `bam_plp_destroy'
/path/samtools/libbam.a(bam_plbuf.o): In function `bam_plbuf_init':
/path/samtools/bam_plbuf.c:45: undefined reference to `bam_plp_init'
/path/samtools/libbam.a(bam_plbuf.o): In function `bam_plbuf_reset':
/path/samtools/bam_plbuf.c:38: undefined reference to `bam_plp_reset'
collect2: ld returned 1 exit status
make: *** [bam2bedgraph] Error 1

I tested already with different samtools versions (1.2, 0.1.17, 0.1.18). I would be happy about any suggestions. Does anybody have an idea?

Best
Robby

[GenoMax]

Have you done the following for your samtools directory?

set the environment variable SAMTOOLS to point to this directory

[Robby]

Yes, I applied the following command

export SAMTOOLS=$SAMTOOLS:/path/samtools

[GenoMax]

Can you try

$ SAMTOOLS='/path/samtools'
$ echo $SAMTOOLS

[Robby]

Thanks for your prompt answers!

I tested your comand first with samtools 0.1.17 and afterwards with samtools 1.2:

$ SAMTOOLS='/path/samtools-0.1.17'
$ echo $SAMTOOLS
/path/software/samtools-0.1.17

For samtools 1.2 I received the same error message mentioned above. For samtools 0.1.17 the error message is slightly different, but probably caused by the same problem:

perl Build.PL --install_base /home/$User/perl5/
Found /path/samtools-0.1.17/bam.h and /path/samtools-0.1.17/libbam.a.
Created MYMETA.yml and MYMETA.json
Creating new 'Build' script for 'Bio-SamTools' version '1.41'

Building Bio-SamTools
which: no nmake in ([...])
gcc -g -Wall -O2 -fPIC -o bam2bedgraph bam2bedgraph.o  -L/path/samtools-0.1.17 -lbam -lpthread -lm -lz
bam2bedgraph.o: In function `main':
/path/Bio-SamTools-1.41/c_bin/bam2bedgraph.c:62: undefined reference to `hts_idx_load'
bam2bedgraph.o: In function `bam_index_destroy':
/path/samtools-1.2/bam.h:449: undefined reference to `hts_idx_destroy'
collect2: ld returned 1 exit status
make: *** [bam2bedgraph] Error 1

For some reason the bam.h from samtools 1.2 is used, although it should point to samtools 1.2.

[GenoMax]

According to the Bio-Samtools README

This is a Perl interface to the SAMtools sequence alignment
interface. It ONLY works on versions of Samtools up to 0.1.19. It does
not work on version 1.0 or higher due to major changes in the library
structure.

so this will not work with new samtools (1.2).

Can you re-try (in a new terminal window) making sure the SAMTOOLS variable is pointing to the older version? Also start with a freshly uncompressed source directory or do a "make clean" to remove any past traces.

[Robby]

OK, that seems to work. I have still some warnings, but at least no errors. So I will start to test, if it really works. Thanks a lot for your help

Building Bio-SamTools
gcc -I/path/samtools-0.1.19 -Ic_bin -I/usr/lib64/perl5/CORE -fPIC -D_IOLIB=2 -D_FILE_OFFSET_BITS=64 -Wformat=0 -c -D_REENTRANT -D_GNU_SOURCE -fno-strict-aliasing -pipe -fstack-protector -I/usr/local/include -D_LARGEFILE_SOURCE -D_FILE_OFFSET_BITS=64 -O2 -g -pipe -Wall -Wp,-D_FORTIFY_SOURCE=2 -fexceptions -fstack-protector --param=ssp-buffer-size=4 -m64 -mtune=generic -o c_bin/bam2bedgraph.o c_bin/bam2bedgraph.c
gcc -I/path/samtools-0.1.19 -Ic_bin -I/usr/lib64/perl5/CORE -DXS_VERSION="1.41" -DVERSION="1.41" -fPIC -D_IOLIB=2 -D_FILE_OFFSET_BITS=64 -Wformat=0 -c -D_REENTRANT -D_GNU_SOURCE -fno-strict-aliasing -pipe -fstack-protector -I/usr/local/include -D_LARGEFILE_SOURCE -D_FILE_OFFSET_BITS=64 -O2 -g -pipe -Wall -Wp,-D_FORTIFY_SOURCE=2 -fexceptions -fstack-protector --param=ssp-buffer-size=4 -m64 -mtune=generic -o lib/Bio/DB/Sam.o lib/Bio/DB/Sam.c
lib/Bio/DB/Sam.xs: In function ‘invoke_pileup_callback_fun’:
lib/Bio/DB/Sam.xs:118: warning: unused variable ‘pileups’
lib/Bio/DB/Sam.xs:116: warning: unused variable ‘pileup_obj’
lib/Bio/DB/Sam.c: In function ‘XS_Bio__DB__Bam_open’:
lib/Bio/DB/Sam.c:563: warning: unused variable ‘packname’
lib/Bio/DB/Sam.c: In function ‘XS_Bio__DB__Bam_index_build’:
lib/Bio/DB/Sam.c:623: warning: unused variable ‘packname’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam_sort_core’:
lib/Bio/DB/Sam.xs:324: warning: implicit declaration of function ‘bam_sort_core’
lib/Bio/DB/Sam.c:647: warning: unused variable ‘packname’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Alignment_qseq’:
lib/Bio/DB/Sam.xs:518: warning: operation on ‘seq’ may be undefined
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Alignment__qscore’:
lib/Bio/DB/Sam.xs:532: warning: pointer targets in passing argument 2 of ‘Perl_newSVpv’ differ in signedness
/usr/lib64/perl5/CORE/proto.h:2210: note: expected ‘const char *’ but argument is of type ‘uint8_t *’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Alignment_aux’:
lib/Bio/DB/Sam.xs:615: warning: pointer targets in passing argument 2 of ‘strncat’ differ in signedness
/usr/include/bits/string3.h:149: note: expected ‘const char * __restrict__’ but argument is of type ‘uint8_t *’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Alignment_aux_keys’:
lib/Bio/DB/Sam.xs:687: warning: pointer targets in passing argument 2 of ‘Perl_newSVpv’ differ in signedness
/usr/lib64/perl5/CORE/proto.h:2210: note: expected ‘const char *’ but argument is of type ‘uint8_t *’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Alignment_data’:
lib/Bio/DB/Sam.xs:710: warning: pointer targets in assignment differ in signedness
lib/Bio/DB/Sam.xs:713: warning: pointer targets in passing argument 2 of ‘Perl_newSVpv’ differ in signedness
/usr/lib64/perl5/CORE/proto.h:2210: note: expected ‘const char *’ but argument is of type ‘uint8_t *’
lib/Bio/DB/Sam.xs: In function ‘XS_Bio__DB__Bam__Header_view1’:
lib/Bio/DB/Sam.xs:916: warning: implicit declaration of function ‘bam_view1’
lib/Bio/DB/Sam.c: In function ‘XS_Bio__DB__Bam__Index_coverage’:
lib/Bio/DB/Sam.xs:1001: warning: unused variable ‘cov’
lib/Bio/DB/Sam.c: In function ‘invoke_pileup_callback_fun’:
lib/Bio/DB/Sam.xs:151: warning: control reaches end of non-void function
ExtUtils::Mkbootstrap::Mkbootstrap('blib/arch/auto/Bio/DB/Sam/Sam.bs')
gcc -shared -O2 -g -pipe -Wall -Wp,-D_FORTIFY_SOURCE=2 -fexceptions -fstack-protector --param=ssp-buffer-size=4 -m64 -mtune=generic -o blib/arch/auto/Bio/DB/Sam/Sam.so lib/Bio/DB/Sam.o c_bin/bam2bedgraph.o -L/path/samtools-0.1.19 -lbam -lpthread -lz
which: no nmake in ([...])
gcc -g -Wall -O2 -fPIC -o bam2bedgraph bam2bedgraph.o  -L/path/samtools-0.1.19 -lbam -lpthread -lm -lz