rsem-calculate-expression: 0 expected read counts
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pathunk
Apr 25, 2012, 6:17:25 PM4/25/12
to RSEM Users
Hi rsem community,
When I run rsem-calculate-expression, the expected counts for nearly
all my 'genes' (contigs) are 0. The very few that are not 0 are very
low numbers. This is puzzling since the reference was assembled with
the same libraries that I gave to rsem to calculate expression. All 6
paired-end libraries are experimental expression assays from the same
genotype.
I built a de novo transcriptome assembly using Abyss. The assembly has
261,872 contigs and N50 = 1389. The assembly is rough as indicated by
the very large number of contigs, but I would still expect that an
appreciable fraction of reads would align. Yet rsem's output reports:
/Volumes/pichia/aman/bowtie-0.12.7/bowtie -q --phred33-quals -n 2 -e
99999999 -l 25 -I 1 -X 1000 -p 4 -a -m 200 -S k76_nn/rsem_k76_nn/
k76_nn_ref -1
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
-2
rp_asg105.fa,rp_asg106.fa,rp_asg107.fa,rp_asg108.fa,rp_asg109.fa,rp_asg110.fa
| gzip > k76_nn_rsem.sam.gz
# reads processed: 55173258
# reads with at least one reported alignment: 6872 (0.01%)
# reads that failed to align: 55166386 (99.99%)
Reported 106235 paired-end alignments to 1 output stream(s)
Looking over the posts in this group, others seem to have had this
problem but I couldn't find a solution. Any ideas on what's going
wrong? Could this be a problem with bowtie? For other purposes I've
been using bowtie2 for alignments since it's recommended for longer
read lengths (mine are 100bp reads).
My commands:
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-prepare-reference --no-
polyA --bowtie-path /Volumes/pichia/aman/bowtie-0.12.7 Ua_76_nn-
contigs.fa k76_nn_ref
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7 --paired-end
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
rp_asg105.fa,rp_asg106.fa,rp_asg107.fa,rp_asg108.fa,rp_asg109.fa,rp_asg110.fa
k76_nn/rsem_k76_nn/k76_nn_ref k76_nn_rsem &
Here's the first few lines of my output file,
k76_nn_rsem.genes.results:
1 0.00 0 0 4.72016114363459e-05 1
10 0.00 0 0 5.76845577305948e-06 10
100 0.00 0 0 0 100
1000 0.00 0 0 1.80834669529904e-05 1000
10000 0.00 0 0 0 10000
100000 0.00 0 0 0 100000
100001 0.00 0 0 0 100001
100002 0.00 0 0 0 100002
100004 0.00 0 0 0 100004
100005 0.00 0 0 0 100005
100007 0.00 0 0 0 100007
100008 0.00 0 0 0 100008
100009 0.00 0 0 0 100009
10001 0.00 0 0 3.05669191568949e-06 10001
100010 0.00 0 0 0 100010
100013 0.00 0 0 0 100013
k76_nn_rsem.isoforms.results begins the same way and is nearly
identical, as expected given that this is a raw unannotated assembly.
When I run rsem-calculate-expression, the expected counts for nearly
all my 'genes' (contigs) are 0. The very few that are not 0 are very
low numbers. This is puzzling since the reference was assembled with
the same libraries that I gave to rsem to calculate expression. All 6
paired-end libraries are experimental expression assays from the same
genotype.
I built a de novo transcriptome assembly using Abyss. The assembly has
261,872 contigs and N50 = 1389. The assembly is rough as indicated by
the very large number of contigs, but I would still expect that an
appreciable fraction of reads would align. Yet rsem's output reports:
/Volumes/pichia/aman/bowtie-0.12.7/bowtie -q --phred33-quals -n 2 -e
99999999 -l 25 -I 1 -X 1000 -p 4 -a -m 200 -S k76_nn/rsem_k76_nn/
k76_nn_ref -1
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
-2
rp_asg105.fa,rp_asg106.fa,rp_asg107.fa,rp_asg108.fa,rp_asg109.fa,rp_asg110.fa
| gzip > k76_nn_rsem.sam.gz
# reads processed: 55173258
# reads with at least one reported alignment: 6872 (0.01%)
# reads that failed to align: 55166386 (99.99%)
Reported 106235 paired-end alignments to 1 output stream(s)
Looking over the posts in this group, others seem to have had this
problem but I couldn't find a solution. Any ideas on what's going
wrong? Could this be a problem with bowtie? For other purposes I've
been using bowtie2 for alignments since it's recommended for longer
read lengths (mine are 100bp reads).
My commands:
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-prepare-reference --no-
polyA --bowtie-path /Volumes/pichia/aman/bowtie-0.12.7 Ua_76_nn-
contigs.fa k76_nn_ref
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7 --paired-end
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
rp_asg105.fa,rp_asg106.fa,rp_asg107.fa,rp_asg108.fa,rp_asg109.fa,rp_asg110.fa
k76_nn/rsem_k76_nn/k76_nn_ref k76_nn_rsem &
Here's the first few lines of my output file,
k76_nn_rsem.genes.results:
1 0.00 0 0 4.72016114363459e-05 1
10 0.00 0 0 5.76845577305948e-06 10
100 0.00 0 0 0 100
1000 0.00 0 0 1.80834669529904e-05 1000
10000 0.00 0 0 0 10000
100000 0.00 0 0 0 100000
100001 0.00 0 0 0 100001
100002 0.00 0 0 0 100002
100004 0.00 0 0 0 100004
100005 0.00 0 0 0 100005
100007 0.00 0 0 0 100007
100008 0.00 0 0 0 100008
100009 0.00 0 0 0 100009
10001 0.00 0 0 3.05669191568949e-06 10001
100010 0.00 0 0 0 100010
100013 0.00 0 0 0 100013
k76_nn_rsem.isoforms.results begins the same way and is nearly
identical, as expected given that this is a raw unannotated assembly.
Colin Dewey
Apr 27, 2012, 2:15:45 PM4/27/12
to rsem-...@googlegroups.com
Hi pathunk,
I noticed that you have paired-end reads. One thing you might check is whether your assembler is using the paired-end data to scaffold your contigs, or if it is simply returning just the contigs. RSEM requires that both ends of a read pair align to the *same transcript/contig sequence* in order for that read pair to have a valid alignment. If your assembly is not scaffolded, then many of your read pairs will not have a valid alignment according to this criterion.
If you can only get contigs, then for quantification with RSEM, you will need to only use one end of your paired-end reads (i.e., treat one end of your paired-end reads as single-end read data). For example, try:
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7 fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
Also, no need to use bowtie2 here. RSEM uses bowtie (v1) in a customized way for optimal quantification and has no problem with longer reads (with the exception that indels are currently not supported).
Best,
Colin
I noticed that you have paired-end reads. One thing you might check is whether your assembler is using the paired-end data to scaffold your contigs, or if it is simply returning just the contigs. RSEM requires that both ends of a read pair align to the *same transcript/contig sequence* in order for that read pair to have a valid alignment. If your assembly is not scaffolded, then many of your read pairs will not have a valid alignment according to this criterion.
If you can only get contigs, then for quantification with RSEM, you will need to only use one end of your paired-end reads (i.e., treat one end of your paired-end reads as single-end read data). For example, try:
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
Also, no need to use bowtie2 here. RSEM uses bowtie (v1) in a customized way for optimal quantification and has no problem with longer reads (with the exception that indels are currently not supported).
Best,
Colin
pathunk
May 5, 2012, 9:59:22 AM5/5/12
to RSEM Users
Hi Colin,
Thanks for your input. As you suggested, I ran calculate-expression
using only my forward reads, treating them as single-end. 86% of the
reads aligned, a big improvement. But rsem is getting stuck at rsem-
run-em. I searched for threads addressing this problem, but couldn't
find anything that's likely to apply to me. When I ran this version of
rsem with paired-end reads, I didn't get stuck at this step (though I
had close to 0% of reads aligning).
command:
rsem-run-em runs indefinitely. When I kill it (>24 hours after
initiation), log reads:
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp k76_nn_rsem_fp -p 4 -b s
k76_nn_rsem_fp.sam.gz 0 --gibbs-out
"/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp k76_nn_rsem_fp -p 4 -b s
k76_nn_rsem_fp.sam.gz 0 --gibbs-out" failed! Plase check if you
provide correct parameters/options for the pipeline!
Thanks for your input. As you suggested, I ran calculate-expression
using only my forward reads, treating them as single-end. 86% of the
reads aligned, a big improvement. But rsem is getting stuck at rsem-
run-em. I searched for threads addressing this problem, but couldn't
find anything that's likely to apply to me. When I ran this version of
rsem with paired-end reads, I didn't get stuck at this step (though I
had close to 0% of reads aligning).
command:
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
k76_nn/rsem_k76_nn/k76_nn_ref k76_nn_rsem_fp &
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa,fp_asg109.fa,fp_asg110.fa
rsem-run-em runs indefinitely. When I kill it (>24 hours after
initiation), log reads:
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp k76_nn_rsem_fp -p 4 -b s
k76_nn_rsem_fp.sam.gz 0 --gibbs-out
"/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp k76_nn_rsem_fp -p 4 -b s
k76_nn_rsem_fp.sam.gz 0 --gibbs-out" failed! Plase check if you
provide correct parameters/options for the pipeline!
b...@cs.wisc.edu
May 5, 2012, 2:29:49 PM5/5/12
to rsem-...@googlegroups.com
Hi Pathunk,
Is there any intermediate output generated by rsem-run-em?
Thanks,
Bo
Is there any intermediate output generated by rsem-run-em?
Thanks,
Bo
pathunk
May 5, 2012, 8:01:38 PM5/5/12
to RSEM Users
Hi Bo, I am not sure which if any are from rsem-run-em, but these are
the folders and files I get:
k76_nn_rsem_fp.sam.gz (12.2gb)
k76_nn_rsem_fp.stat (folder)
k76_nn_rsem_fp.cnt (4kb)
k76_nn_rsem_fp_run3.temp (folder)
k76_nn_rsem_fp_alignable.fq (7.6 gb)
k76_nn_rsem_fp_alignable.fq.ridx (148mb)
k76_nn_rsem_fp_un.fq (1.22gb)
k76_nn_rsem_fp.dat (3.88gb)
k76_nn_rsem_fp.mparams (4kb)
the folders and files I get:
k76_nn_rsem_fp.sam.gz (12.2gb)
k76_nn_rsem_fp.stat (folder)
k76_nn_rsem_fp.cnt (4kb)
k76_nn_rsem_fp_run3.temp (folder)
k76_nn_rsem_fp_alignable.fq (7.6 gb)
k76_nn_rsem_fp_alignable.fq.ridx (148mb)
k76_nn_rsem_fp_un.fq (1.22gb)
k76_nn_rsem_fp.dat (3.88gb)
k76_nn_rsem_fp.mparams (4kb)
b...@cs.wisc.edu
May 5, 2012, 8:17:37 PM5/5/12
to rsem-...@googlegroups.com
Hi Pathunk,
Can you find a "nohup.out" file?
Best,
Bo
Can you find a "nohup.out" file?
Best,
Bo
pathunk
May 6, 2012, 10:19:20 AM5/6/12
to RSEM Users
Hi Bo, yes there's also a nohup.out file. I pasted the tail end of the
output from that file earlier, but here's the full contents.
/Volumes/pichia/aman/bowtie-0.12.7/bowtie -q --phred33-quals -n 2 -e
99999999 -l 25 -p 4 -a -m 200 -S k76_nn/rsem_k76_nn/k76_nn_ref
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa | gzip >
k76_nn_rsem_fp_run3.sam.gz
# reads processed: 40670279
# reads with at least one reported alignment: 35052287 (86.19%)
# reads that failed to align: 5257258 (12.93%)
# reads with alignments suppressed due to -m: 360734 (0.89%)
Reported 386736847 alignments to 1 output stream(s)
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-parse-alignments k76_nn/
rsem_k76_nn/k76_nn_ref k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 s
k76_nn_rsem_fp_run3.sam.gz -t 1 -tag non-unique
[samopen] SAM header is present: 261872 sequences.
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Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-build-read-index 32 1 0
k76_nn_rsem_fp_run3.temp/k76_nn_rsem_fp_run3_alignable.fq
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Build Index k76_nn_rsem_fp_run3.temp/k76_nn_rsem_fp_run3_alignable.fq
is Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 -p 4 -
b s k76_nn_rsem_fp_run3.sam.gz 0 --gibbs-out
"/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 -p 4 -
b s k76_nn_rsem_fp_run3.sam.gz 0 --gibbs-out" failed! Plase check if
output from that file earlier, but here's the full contents.
/Volumes/pichia/aman/bowtie-0.12.7/bowtie -q --phred33-quals -n 2 -e
fp_asg105.fa,fp_asg106.fa,fp_asg107.fa,fp_asg108.fa | gzip >
k76_nn_rsem_fp_run3.sam.gz
# reads processed: 40670279
# reads with at least one reported alignment: 35052287 (86.19%)
# reads that failed to align: 5257258 (12.93%)
# reads with alignments suppressed due to -m: 360734 (0.89%)
Reported 386736847 alignments to 1 output stream(s)
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-parse-alignments k76_nn/
rsem_k76_nn/k76_nn_ref k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 s
k76_nn_rsem_fp_run3.sam.gz -t 1 -tag non-unique
[samopen] SAM header is present: 261872 sequences.
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Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-build-read-index 32 1 0
k76_nn_rsem_fp_run3.temp/k76_nn_rsem_fp_run3_alignable.fq
FIN 1000000
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Build Index k76_nn_rsem_fp_run3.temp/k76_nn_rsem_fp_run3_alignable.fq
is Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 -p 4 -
b s k76_nn_rsem_fp_run3.sam.gz 0 --gibbs-out
"/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run3 k76_nn_rsem_fp_run3 -p 4 -
b s k76_nn_rsem_fp_run3.sam.gz 0 --gibbs-out" failed! Plase check if
you provide correct parameters/options for the pipeline!
b...@cs.wisc.edu
May 6, 2012, 11:49:52 AM5/6/12
to rsem-...@googlegroups.com
Hi Pathunk,
That's really strange. Can you try to only use one reads file (say
fp_asg105.fa) and see if RSEM can finish?
Best,
Bo
That's really strange. Can you try to only use one reads file (say
fp_asg105.fa) and see if RSEM can finish?
Best,
Bo
pathunk
May 7, 2012, 9:52:37 AM5/7/12
to RSEM Users
Hi Bo, no luck. I started a run using a single read file more than 20
hours ago, and it's still on rsem-run-em (I didn't kill the process
this time; it's still running). nohup.out looks basically the same as
before, and so does the set of intermediate files created. The
nohup.out file was last modified about 2 hours after I initiated rsem-
calculate-expression.
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
pichia/aman/bowtie-0.12.7 fp_asg105.fa k76_nn/rsem_k76_nn/k76_nn_ref
k76_nn_rsem_fp_run4 &
nohup.out:
# reads processed: 11133404
# reads with at least one reported alignment: 9566220 (85.92%)
# reads that failed to align: 1477080 (13.27%)
# reads with alignments suppressed due to -m: 90104 (0.81%)
Reported 104810892 alignments to 1 output stream(s)
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-parse-alignments k76_nn/
rsem_k76_nn/k76_nn_ref k76_nn_rsem_fp_run4 k76_nn_rsem_fp_run4 s
k76_nn_rsem_fp_run4.sam.gz -t 1 -tag non-unique
is Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run4 k76_nn_rsem_fp_run4 -p 4 -
b s k76_nn_rsem_fp_run4.sam.gz 0 --gibbs-out
> > Parsed 324000000 entries...
>
> read more »
hours ago, and it's still on rsem-run-em (I didn't kill the process
this time; it's still running). nohup.out looks basically the same as
before, and so does the set of intermediate files created. The
nohup.out file was last modified about 2 hours after I initiated rsem-
calculate-expression.
nohup /Volumes/pichia/aman/rsem-1.1.18-modified/rsem-calculate-
expression -p 4 --calc-ci --tag non-unique --bowtie-path /Volumes/
k76_nn_rsem_fp_run4 &
nohup.out:
/Volumes/pichia/aman/bowtie-0.12.7/bowtie -q --phred33-quals -n 2 -e
99999999 -l 25 -p 4 -a -m 200 -S k76_nn/rsem_k76_nn/k76_nn_ref
fp_asg105.fa | gzip > k76_nn_rsem_fp_run4.sam.gz
99999999 -l 25 -p 4 -a -m 200 -S k76_nn/rsem_k76_nn/k76_nn_ref
# reads processed: 11133404
# reads with at least one reported alignment: 9566220 (85.92%)
# reads that failed to align: 1477080 (13.27%)
# reads with alignments suppressed due to -m: 90104 (0.81%)
Reported 104810892 alignments to 1 output stream(s)
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-parse-alignments k76_nn/
rsem_k76_nn/k76_nn_ref k76_nn_rsem_fp_run4 k76_nn_rsem_fp_run4 s
k76_nn_rsem_fp_run4.sam.gz -t 1 -tag non-unique
Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-build-read-index 32 1 0
k76_nn_rsem_fp_run4.temp/k76_nn_rsem_fp_run4_alignable.fq
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-build-read-index 32 1 0
FIN 1000000
FIN 2000000
FIN 3000000
FIN 4000000
FIN 5000000
FIN 6000000
FIN 7000000
FIN 8000000
FIN 9000000
Build Index k76_nn_rsem_fp_run4.temp/k76_nn_rsem_fp_run4_alignable.fq
FIN 2000000
FIN 3000000
FIN 4000000
FIN 5000000
FIN 6000000
FIN 7000000
FIN 8000000
FIN 9000000
is Done!
/Volumes/pichia/aman/rsem-1.1.18-modified/rsem-run-em k76_nn/
rsem_k76_nn/k76_nn_ref 1 k76_nn_rsem_fp_run4 k76_nn_rsem_fp_run4 -p 4 -
b s k76_nn_rsem_fp_run4.sam.gz 0 --gibbs-out
>
> read more »
b...@cs.wisc.edu
May 7, 2012, 10:34:22 AM5/7/12
to rsem-...@googlegroups.com
Hi Pathunk,
Two things. 1) Can you check if
k76_nn/rsem_k76_nn/k76_nn_ref[.seq/.ti/.grp] exist? 2) Why do you want to
set --tag option?
Best,
Bo
>> read more �
>
Two things. 1) Can you check if
k76_nn/rsem_k76_nn/k76_nn_ref[.seq/.ti/.grp] exist? 2) Why do you want to
set --tag option?
Best,
Bo
>
pathunk
May 7, 2012, 11:09:38 AM5/7/12
to RSEM Users
Hi Bo,
Those files do exist. Here's the contents of k76_nn/rsem_k76_nn/
k76_nn_ref.1.ebwt
k76_nn_ref.2.ebwt
k76_nn_ref.3.ebwt
k76_nn_ref.4.ebwt
k76_nn_ref.chrlist
k76_nn_ref.grp
k76_nn_ref.idx.fa
k76_nn_ref.rev.1.ebwt
k76_nn_ref.rev.2.ebwt
k76_nn_ref.seq
k76_nn_ref.ti
k76_nn_ref.transcripts.fa
I thought I'd set the tag option since I have not yet thoroughly
evaluated or processed my assembly (this is a de novo transcriptome
assembly with 261,872 contigs based on the same read libraries for
which I'd like to calculate expression differences) and I was worried
that some reads might align to a large number of contigs. As I
understand the tag option, it flags reads that have "too many valid
alignments."
Could the large number of contigs be an issue here?
> >> > Parsed 129000000 entries...
>
> read more »
Those files do exist. Here's the contents of k76_nn/rsem_k76_nn/
k76_nn_ref.1.ebwt
k76_nn_ref.2.ebwt
k76_nn_ref.3.ebwt
k76_nn_ref.4.ebwt
k76_nn_ref.chrlist
k76_nn_ref.grp
k76_nn_ref.idx.fa
k76_nn_ref.rev.1.ebwt
k76_nn_ref.rev.2.ebwt
k76_nn_ref.seq
k76_nn_ref.ti
k76_nn_ref.transcripts.fa
I thought I'd set the tag option since I have not yet thoroughly
evaluated or processed my assembly (this is a de novo transcriptome
assembly with 261,872 contigs based on the same read libraries for
which I'd like to calculate expression differences) and I was worried
that some reads might align to a large number of contigs. As I
understand the tag option, it flags reads that have "too many valid
alignments."
Could the large number of contigs be an issue here?
>
> read more »
b...@cs.wisc.edu
May 7, 2012, 12:44:05 PM5/7/12
to rsem-...@googlegroups.com
Hi Pathunk,
I do not think so. Please try these, take the header and first 1000 reads
in the .sam.gz file (let us call it truncated sam file) generated and see
if RSEM can finish it in time. If not, please send me the truncated sam
file and k76_nn_ref.transcripts.fa. I'll see what happens.
Thanks,
Bo
>> read more �
>
I do not think so. Please try these, take the header and first 1000 reads
in the .sam.gz file (let us call it truncated sam file) generated and see
if RSEM can finish it in time. If not, please send me the truncated sam
file and k76_nn_ref.transcripts.fa. I'll see what happens.
Thanks,
Bo
>
pathunk
May 15, 2012, 3:51:04 PM5/15/12
to RSEM Users
Hi Bo,
I created a truncated sam file by concatenating the entire header
section with the first 1000 lines of the sequence portion of the file.
When I ran rsem-calculate-expression, I did get isoforms.results and
genes.results files, but again nearly all of the expected read counts
were 0 (this was the original problem I posted to initiate this
thread).
When I looked line-by-line at the truncated sam file, I noticed that
many of the contigs were short, and contained duplicated regions
relative to other contigs. In case this presented a problem, I tried a
second run, where I filtered my original reference fasta (i.e. my de
novo assembly) to retain only those sequences >1000nt. I ran rsem-
prepare-reference on this filtered reference, and then ran calculate-
expression. Again, rsem-run-em ran forever. I then went through the
same truncation procedure. When I ran calculate-expression with the
new truncated sam, the .results files were created, but again ~99% of
'genes' showed 0 expected counts, and the other ~1% had 1 expected
alignment.
The reference was created with these same sets of reads, so it is
strange to me that I would have so few expected counts. Though I
didn't see alignment stats in the output from the run with the
truncated sam, the run with the full (>1000nt) filtered reference
showed that 24 million reads aligned:
# reads processed: 61349468
# reads with at least one reported alignment: 24294996 (39.60%)
# reads that failed to align: 37025542 (60.35%)
# reads with alignments suppressed due to -m: 28930 (0.05%)
I will send you the filtered truncated sam and the ref.transcripts.fa
file in case you'd like to look at them. Please let me know if you
have any thoughts.
Thanks,
Aman (pathunk)
> >> >> > Parsed 65000000 entries...
>
> read more »
I created a truncated sam file by concatenating the entire header
section with the first 1000 lines of the sequence portion of the file.
When I ran rsem-calculate-expression, I did get isoforms.results and
genes.results files, but again nearly all of the expected read counts
were 0 (this was the original problem I posted to initiate this
thread).
When I looked line-by-line at the truncated sam file, I noticed that
many of the contigs were short, and contained duplicated regions
relative to other contigs. In case this presented a problem, I tried a
second run, where I filtered my original reference fasta (i.e. my de
novo assembly) to retain only those sequences >1000nt. I ran rsem-
prepare-reference on this filtered reference, and then ran calculate-
expression. Again, rsem-run-em ran forever. I then went through the
same truncation procedure. When I ran calculate-expression with the
new truncated sam, the .results files were created, but again ~99% of
'genes' showed 0 expected counts, and the other ~1% had 1 expected
alignment.
The reference was created with these same sets of reads, so it is
strange to me that I would have so few expected counts. Though I
didn't see alignment stats in the output from the run with the
truncated sam, the run with the full (>1000nt) filtered reference
showed that 24 million reads aligned:
# reads processed: 61349468
# reads with at least one reported alignment: 24294996 (39.60%)
# reads that failed to align: 37025542 (60.35%)
# reads with alignments suppressed due to -m: 28930 (0.05%)
I will send you the filtered truncated sam and the ref.transcripts.fa
file in case you'd like to look at them. Please let me know if you
have any thoughts.
Thanks,
Aman (pathunk)
>
> read more »
b...@cs.wisc.edu
May 27, 2012, 2:22:18 PM5/27/12
to rsem-...@googlegroups.com
Hi Aman,
Sorry for late reply.
For the truncated data, you only have 388 reads which can align to your
reference. So it is normal that you find most of contigs have expected
count 0.
I cannot understand why RSEM stuck on your large dataset while it can pass
this small one.
But there is still things we can check. First, for the large data set,
have you seen some outputs like below:
Refs.loadRefs finished!
Thread 0 : N = 49, NHit = 53
Thread 1 : N = 47, NHit = 53
Thread 2 : N = 45, NHit = 53
Thread 3 : N = 47, NHit = 53
Thread 4 : N = 46, NHit = 53
Thread 5 : N = 49, NHit = 53
Thread 6 : N = 48, NHit = 53
DAT 0 reads left
Thread 7 : N = 57, NHit = 59
EM_init finished!
estimateFromReads, N0 finished.
estimateFromReads, N1 finished.
estimateFromReads, N2 finished.
In addition, can you send me the sample.cnt file under sample.temp directory?
Thanks,
Bo
>> read more �
>
Sorry for late reply.
For the truncated data, you only have 388 reads which can align to your
reference. So it is normal that you find most of contigs have expected
count 0.
I cannot understand why RSEM stuck on your large dataset while it can pass
this small one.
But there is still things we can check. First, for the large data set,
have you seen some outputs like below:
Refs.loadRefs finished!
Thread 0 : N = 49, NHit = 53
Thread 1 : N = 47, NHit = 53
Thread 2 : N = 45, NHit = 53
Thread 3 : N = 47, NHit = 53
Thread 4 : N = 46, NHit = 53
Thread 5 : N = 49, NHit = 53
Thread 6 : N = 48, NHit = 53
DAT 0 reads left
Thread 7 : N = 57, NHit = 59
EM_init finished!
estimateFromReads, N0 finished.
estimateFromReads, N1 finished.
estimateFromReads, N2 finished.
In addition, can you send me the sample.cnt file under sample.temp directory?
Thanks,
Bo
>
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