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#1 createSNPbedfile.sh
closed
2018-09-25
2018-09-20
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Hi
I am trying to use Allelome pro. I I trying to use the helperscript createSNPbedfile.sh.
Could you tell me if I have to write in the strain name or the corresponding number in the list given?
The two mouse strains we are using includes C57Bl/6J which is the reference and not given as a choice of strain.
many thanks
Hi Carol,
Only the numbers are required. Just use the number 8 for C57Bl/6J which is
listed as C57Bl/6NJ. These two strains should be genetically identically,
the only difference is that they were hosted in different mouse facilities
N for NIH and J for Jackson lab.
command:
sh createSNPbedfile.sh path/mgp.v3.snps.rsIDdbSNPv137.vcf outputfile.bed
Also remember the order of the strains you use to generate the SNP file,
which is important later in the Allelome.PRO pipeline when you fill out the
configs! I would recommend to run first the files in the test_run folder
(provided in the Alleome.PRO folder) which helps a lot to understand which
inputs are required and to test if all the required tools (bedtools
samtools R etc) are loaded correctly in your system.
Good luck and let me know if you need more help!
All the best,
Daniel
Am Do., 20. Sep. 2018 um 04:43 Uhr schrieb Carol Edwards cae28@users.sourceforge.net:
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#1Dear Daniel,
Thanks for your help.
We are trying to run the test data but we keep getting the following
error messages
start read trimming for sample 1
start read trimming for sample 2
start read trimming for sample 3
start read trimming for sample 4
[E::hts_open_format] fail to open file
'./test_run/results//2018_09_21_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/fc1_MEF_CF_1_Igf2r_cl/BAM_trim/trimmed_s.bam'
samtools view: failed to open
"./test_run/results//2018_09_21_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/fc1_MEF_CF_1_Igf2r_cl/BAM_trim/trimmed_s.bam"
for reading: No such file or directory
[E::hts_open_format] fail to open file
'./test_run/results//2018_09_21_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/fc2_MEF_CF_2_Igf2r_cl/BAM_trim/trimmed_s.bam'
samtools view: failed to open
"./test_run/results//2018_09_21_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/fc2_MEF_CF_2_Igf2r_cl/BAM_trim/trimmed_s.bam"
for reading: No such file or directory
And
-e Error:
RNAseq_MEF_XX_E12_5_chr17_12350000_13000000_locus_full.txt not found
check info.txt to see if SNPs were available for the analysis.
analysis.
Our versions of bedtools, samtools and R are all newer than the ones
suggested and are in the PATH environment variable.
Any ideas to why this is not working would be much appreciated
Best wishes
Carol
On 20-09-2018 13:19, daniel andergassen wrote:
--
Carol Edwards PhD,
Department of Genetics
University of Cambridge
Genetics Building
Downing Street
Cambridge CB2 3EH
Phone 44-1223-333981
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#1Hi Carol,
I just run the testdata and got the same error by using samtools (v1.5).
Than I was running the pipeline again using the suggested samtools
version 0.1.19
and it worked!
Don't forget to also load R in your System which is needed later in the
script!
Good luck and let me know if it works!
All the best,
Daniel
Am Fr., 21. Sep. 2018 um 07:06 Uhr schrieb Carol Edwards cae28@users.sourceforge.net:
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#1Thanks Daniel,
we are a bit reluctant to change our version of SAMtools to an earlier
one.
Is there are point in the pipeline where we can specify a different
version of SAMtools rather than using the PATH environments one?
Carol
On 21-09-2018 13:20, daniel andergassen wrote:
--
Carol Edwards PhD,
Department of Genetics
University of Cambridge
Genetics Building
Downing Street
Cambridge CB2 3EH
Phone 44-1223-333981
Related
Tickets:
#1Hi Carol,
We will upload an updated version of Allelome.PRO next week that runs with
the new version of Samtools.
I will update you when you can download it.
Daniel
Am Fr., 21. Sep. 2018 um 09:19 Uhr schrieb Carol Edwards cae28@users.sourceforge.net:
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#1Hi,
Just to clarify: C57Bl/6J and C57Bl/6NJ are not genetically identical - for known differences see https://www.jax.org/strain/005304. However most of the SNPs mapped using C57Bl/6NJ will be identical in C57Bl/6J. Importantly the Allelome.PRO pipeline does identify correctly annotated SNPs by using reciprocal crosses and therefore it is safe to use C57Bl/6NJ for any C57Bl/6 strain.
best,
Florian
Hi Carol,
We fixed the issue with Samtools, just click on the link to download an
updated version of Allelome.PRO that works with the newest version of
samtools (tested for samtools v1.3 and v1.5).
https://sourceforge.net/u/fpauler/allelomepro/ci/master/tarball
All the best,
Daniel
Am Sa., 22. Sep. 2018 um 07:02 Uhr schrieb Florian Pauler fpauler@users.sourceforge.net:
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#1On 23-09-2018 16:46, daniel andergassen wrote:
Dear Daniel,
Thanks for that. The samtools issue has resolved but I still get no SNP
info back from the pipeline.
in the info.txt no SNPs were discovered
Read counts:
Total reads in forward cross replicate 1: 140667
Total reads in forward cross replicate 2: 145102
Total reads in reverse cross replicate 1: 156151
Total reads in reverse cross replicate 2: 153995
Number of reads overlapping at least one SNP in forward cross replicate
1: 0
Number of reads overlapping at least one SNP in forward cross replicate
2: 0
Number of reads overlapping at least one SNP in reverse cross replicate
1: 0
Number of reads overlapping at least one SNP in reverse cross replicate
2: 0
and I am still getting errors at the end
I have pasted the entire output.
Thanks for all your help!
Best wishes
Carol
pipeline location: ./
outputdir: ./test_run/results
annotation: ./test_run/annotation/RefSeq_annotation_Igf2r_cl.bed
snp_file: ./test_run/SNP/SNPs_Igf2r_cl.bed
strains: CAST;FVB
forward cross, replicate 1: ./test_run/samples/MEF_CF_1_Igf2r_cl.bam
forward cross, replicate 2: ./test_run/samples/MEF_CF_2_Igf2r_cl.bam
reverse cross, replicate 1: ./test_run/samples/MEF_FC_1_Igf2r_cl.bam
reverse cross, replicate 2: ./test_run/samples/MEF_FC_2_Igf2r_cl.bam
bamfile sorted: 1
allelic_ratio: 0.7
fdr_param: 1
minreads: 1
Plot_titel: RNAseq_MEF_XX_E12_5_chr17_12350000_13000000
Plot_y_axis: RefSeq_genes
Intersecting SNP file with annotation file to yield a list of the SNPs
used for this analysis including information which genes contain this
SNP
start read trimming for sample 1
start read trimming for sample 2
start read trimming for sample 3
start read trimming for sample 4
creating and formatting the pileup files for each cross
[mpileup] 1 samples in 1 input files
[mpileup] 1 samples in 1 input files
[mpileup] 1 samples in 1 input files
[mpileup] 1 samples in 1 input files
joining the pileup files with the data from the SNP list
handling spliced reads and insertions/deletions
Merge all the pileup files from the 4 crosses to one file
Counting the reads over each SNP for each strain in all four crosses
R script which score the candidates and categorises them as imprinted,
strain biased and biallelic based on the FDR and the ratio set in the
config file
Loading required package: plyr
Loading required package: gplots
Attaching package: âgplotsâ
The following object is masked from âpackage:statsâ:
[1] "----- Start scoring script -----"
[1]
"./test_run/results/2018_09_24_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1"
[2] "./test_run/annotation/RefSeq_annotation_Igf2r_cl.bed"
[3]
"./test_run/results/2018_09_24_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/annotation/annotation_overlapping_snps.txt"
[4]
"./test_run/results/2018_09_24_MEF_CF_1_Igf2r_cl_MEF_CF_2_Igf2r_cl_MEF_FC_1_Igf2r_cl_MEF_FC_2_Igf2r_cl_RefSeq_annotation_Igf2r_cl.bed_1_1/debug/read_count_per_SNP.txt"
[5] "0.7"
[6] "1"
[7] "CAST;FVB"
[8] "RNAseq_MEF_XX_E12_5_chr17_12350000_13000000"
[9] "RefSeq_genes"
[1] "Loading data"
used (Mb) gc trigger (Mb) max used (Mb)
Ncells 304939 16.3 592000 31.7 350000 18.7
Vcells 422670 3.3 1023718 7.9 786430 6.0
[1] "Calculating number of covered SNPs for each Gene in each cross"
[1] "Summing up all the reads over a SNP within a gene"
[1] "Calculating the RPSM (Read per SNP per milion total covered SNP
Reads)"
used (Mb) gc trigger (Mb) max used (Mb)
Ncells 307533 16.5 592000 31.7 383315 20.5
Vcells 427100 3.3 1023718 7.9 786430 6.0
[1] "Calculation of the imprinting SNP score"
Warning messages:
1: In min(x) : no non-missing arguments to min; returning Inf
2: In max(x) : no non-missing arguments to max; returning -Inf
[1] "Calculating the the Imprinted score and strain score"
Warning messages:
1: In min(x) : no non-missing arguments to min; returning Inf
2: In max(x) : no non-missing arguments to max; returning -Inf
[1] "Calculating the maternal/total reads ratio for each transcript &
cross"
[1] "Calculating the strain1/total reads ratio for each transcript &
cross"
[1] "Calculating the maternal/total reads ratio for each transcript &
cross"
[1] "Calculating the strain1/total reads ratio for each transcript &
cross"
[1] "Calculating the FDR for the SNPs"
[1] "Calculating the FDR for the loci"
[1] "Categorising results into MAT,PAT,BAE,strain1 bias,strain2
bias,NI,NS"
[1] "Write output and debug tables"
[1] "Score script finished"
Generate Bedfiles for the UCSC browser
-e Error:
RNAseq_MEF_XX_E12_5_chr17_12350000_13000000_locus_full.txt not found
check info.txt to see if SNPs were available for the analysis.
-e Error: SNP_table_all.txt not found
check info.txt to see if SNPs and/or loci were available for the
analysis.
Run finished
--
Carol Edwards PhD,
Department of Genetics
University of Cambridge
Genetics Building
Downing Street
Cambridge CB2 3EH
Phone 44-1223-333981
Hi Carol,
Just to make sure that your environment is correctly configured:
Have you installed samtools, bedtools and perl on your system?
Can you run intersectbed (from the bedtools suite) from the command line?
best,
Florian
Hi Florian,
we can run bedtools intersect from the command line but intersectbed
does not exist in version 2.26.
we can run intersectBed which I believe is the same as intersect
Carol
On 24-09-2018 11:42, Florian Pauler wrote:
--
Carol Edwards PhD,
Department of Genetics
University of Cambridge
Genetics Building
Downing Street
Cambridge CB2 3EH
Phone 44-1223-333981
Hi Carol,
Sorry for my sloppy typing - of course it is "intersectBed".
Could you provide me the compressed output folder via dropbox or similar ? - in this way I can perhaps trace back the error.
best,
Florian
Hi Florian,
I have hopefully sent you the dropbox link with the file in it
Carol
On 24-09-2018 12:42, Florian Pauler wrote:
--
Carol Edwards PhD,
Department of Genetics
University of Cambridge
Genetics Building
Downing Street
Cambridge CB2 3EH
Phone 44-1223-333981
2 problems were solved in this ticket:
1) SNPs from C57Bl/6JN can be safely used for any C57Bl/6 strain
2) using bedtools 2.27 solved the problem of empty output files