[shi]

And sorting the sam file takes long time. is there any alternative to sort the sam. let me remind you that the sam file size is ~120GB.

If the purpose of your sorting is to count reads for genes, then you do not need to do that if you use featureCounts in Subread package . featureCounts reorders reads to make reads from the same pair be adjacent to each other before counting them. The reordering process is more efficient than sorting.

For more details, have a look at Subread users guide

http://bioinf.wehi.edu.au/subread-pa...UsersGuide.pdf

or featureCounts publication

http://www.ncbi.nlm.nih.gov/pubmed/24227677

[Dinesh Heisnam]

we have to abort 'samtools sort -n' cozit was taking all the drive memory.
we tries the feature count as Shi suggest but get aborted after while. the log is
"
|| ||
|| Load annotation file merged.gtf ... ||
|| Number of features is 61960 ||
|| Number of meta-features is 53522 ||
|| Number of chromosomes is 51872 ||
|| ||
|| Process SAM file accepted_hits_A1.sam... ||
|| Assign fragments (read pairs) to features... ||
|| Each fragment is counted once. ||
|| Found reads that are not properly paired. ||
|| (missing mate or the mate is not the next read) ||
*** glibc detected *** /home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts: free(): invalid next size (fast): 0x000000001aee7a90 ***
======= Backtrace: =========
/lib64/libc.so.6[0x31618750c6]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x4486bd]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x405338]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x409bbd]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x40cbc0]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x40db80]
/lib64/libc.so.6(__libc_start_main+0xfd)[0x316181ecdd]
/home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts[0x401e59]
======= Memory map: ========
00400000-00466000 r-xp 00000000 fd:02 231998440 /home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts
00666000-00667000 rw-p 00066000 fd:02 231998440 /home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts
02658000-1aef6000 rw-p 00000000 00:00 0 [heap]
3161000000-3161020000 r-xp 00000000 fd:00 1715057 /lib64/ld-2.12.so
316121f000-3161220000 r--p 0001f000 fd:00 1715057 /lib64/ld-2.12.so
3161220000-3161221000 rw-p 00020000 fd:00 1715057 /lib64/ld-2.12.so
3161221000-3161222000 rw-p 00000000 00:00 0
3161800000-3161997000 r-xp 00000000 fd:00 1715058 /lib64/libc-2.12.so
3161997000-3161b97000 ---p 00197000 fd:00 1715058 /lib64/libc-2.12.so
3161b97000-3161b9b000 r--p 00197000 fd:00 1715058 /lib64/libc-2.12.so
3161b9b000-3161b9c000 rw-p 0019b000 fd:00 1715058 /lib64/libc-2.12.so
3161b9c000-3161ba1000 rw-p 00000000 00:00 0
3162000000-3162017000 r-xp 00000000 fd:00 1715061 /lib64/libpthread-2.12.so
3162017000-3162216000 ---p 00017000 fd:00 1715061 /lib64/libpthread-2.12.so
3162216000-3162217000 r--p 00016000 fd:00 1715061 /lib64/libpthread-2.12.so
3162217000-3162218000 rw-p 00017000 fd:00 1715061 /lib64/libpthread-2.12.so
3162218000-316221c000 rw-p 00000000 00:00 0
3162400000-3162483000 r-xp 00000000 fd:00 1715059 /lib64/libm-2.12.so
3162483000-3162682000 ---p 00083000 fd:00 1715059 /lib64/libm-2.12.so
3162682000-3162683000 r--p 00082000 fd:00 1715059 /lib64/libm-2.12.so
3162683000-3162684000 rw-p 00083000 fd:00 1715059 /lib64/libm-2.12.so
3162800000-3162815000 r-xp 00000000 fd:00 1715063 /lib64/libz.so.1.2.3
3162815000-3162a14000 ---p 00015000 fd:00 1715063 /lib64/libz.so.1.2.3
3162a14000-3162a15000 r--p 00014000 fd:00 1715063 /lib64/libz.so.1.2.3
3162a15000-3162a16000 rw-p 00015000 fd:00 1715063 /lib64/libz.so.1.2.3
3167c00000-3167c16000 r-xp 00000000 fd:00 1715067 /lib64/libgcc_s-4.4.6-20110824.so.1
3167c16000-3167e15000 ---p 00016000 fd:00 1715067 /lib64/libgcc_s-4.4.6-20110824.so.1
3167e15000-3167e16000 rw-p 00015000 fd:00 1715067 /lib64/libgcc_s-4.4.6-20110824.so.1
7f5a5358d000-7f5a55ca4000 rw-p 00000000 00:00 0
7f5a55ca4000-7f5a55d1b000 rw-p 00000000 00:00 0
7f5a55d1b000-7f5a55d92000 rw-p 00000000 00:00 0
7f5a55dd1000-7f5a55e48000 rw-p 00000000 00:00 0
7f5a55e72000-7f5a56193000 rw-p 00000000 00:00 0
7f5a561bb000-7f5a56235000 rw-p 00000000 00:00 0
7f5a562ce000-7f5a5639a000 rw-p 00000000 00:00 0
7f5a56414000-7f5a56543000 rw-p 00000000 00:00 0
7f5a56599000-7f5a56748000 rw-p 00000000 00:00 0
7f5a56778000-7f5a5677c000 rw-p 00000000 00:00 0
7fff5239a000-7fff523af000 rw-p 00000000 00:00 0 [stack]
7fff523ff000-7fff52400000 r-xp 00000000 00:00 0 [vdso]
ffffffffff600000-ffffffffff601000 r-xp 00000000 00:00 0 [vsyscall]
Aborted (core dumped)
"
we have no idea what went wrong

[shi]

Can you provide your command for running featureCounts? You use version 1.4.3?

[Dinesh Heisnam]

we used the command
" [root@s2x3850x5 sam_files]# /home/gopal/Application/subread-1.4.3-Linux-x86_64/bin/featureCounts -p -T 20 -a merged.gtf -t exon -g gene_id -o counts_A1.txt accepted_hits_A1.sam "

[shi]

We just released a patched version (1.4.3-p1). Could you try that to see if it works for you?