Hello all,
one follow-up question regarding the baits file - I downloaded the file from Agilent earray website (SureSelect Human All Exon 50Mb), however it does not contain the strand info which seem to be needed by Picard's CalculateHsMetrics tool. Did the bait files that other people used had the strand info? If yes, where did they get them? If not, how did they circumvent Picard's requirement? Is the strand info actually used by the tool?
Thanks,
Sanja
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
% reads on target does not equal % bp on target
Originally posted by ECO View PostAnother vote for Picard's CalculateHsMetrics. It's in the public Galaxy (http://main.g2.bx.psu.edu/ under "NGS: Picard (beta)").
Leave a comment:
-
Thanks!
Thank you Jon_Keats! The -H seemed to have done the trick!
chongm
Leave a comment:
-
Can you show a bit of your intervals file?
Some of the header and some of the actual intervals.
Not sure if that is the issue, but its where I usually start.
Leave a comment:
-
Has anyone used hsmetrics for Illumina truseq exome targets? I'm having issues running this (even though I'm using the same modified bed file - with header from samtools - for both target and bait intervals).
Here is what I've done
samtools view -h sample.bam > header.txt
awk -F $'\t' 'BEGIN { OFS = FS } {print $1,$2,$3,$6,$4;}' truseq.bed > intervals
cat header.txt intervals > truseq_intervals
java -Xmx4g -Djava.io.tmpdir=/tmp/ \
-jar CalculateHsMetrics.jar \
INPUT=sample.bam \
OUTPUT=sample.hsmetrics \
TARGET_INTERVALS=truseq_intervals\
BAIT_INTERVALS=truseq_intervals
Exception in thread "main" net.sf.picard.PicardException: Invalid interval record contains 22 fields: MISEQ:23:000000000-A34AH:1:1111:11133:10035 99 chrM 339 60 151M = 469 283 ACACATCTCTGCCAAACCCCAAAAACAAAGAACCCTAACACCAGCCTAACCAGATTTCAAATTTTATCTTTTGGCGGTATGCACTTTTAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCA AAAAABBBDDDDDDEEGGGGGGIIIHIIIIIIIIHIIIIIIHIIIIIIIIIIIIIIIIIIHHIIIIIIIIIIIHHHHEEHIIHHHHHHHHHHHHHHHHFHHGGGGHGGGGGGGGGGGGGGEGGGGGGGGGGGGGEGGGGGGEGGGGGGGGG X0:i:1 X1:i:0 MD:Z:71A79 RG:Z:Exome_2011-002 XG:i:0 AM:i:37 NM:i:1 SM:i:37 XM:i:1XO:i:0 XT:A:U
at net.sf.picard.util.IntervalList.fromReader(IntervalList.java:209)
at net.sf.picard.util.IntervalList.fromFile(IntervalList.java:169)
at net.sf.picard.analysis.directed.CollectTargetedMetrics.doWork(CollectTargetedMetrics.java:99)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
at net.sf.picard.analysis.directed.CalculateHsMetrics.main(CalculateHsMetrics.java:73)
Leave a comment:
-
Originally posted by bwubb View PostSo this method more or less worked. Picard seems to demand the columns be tab separated so my awk was more like:
awk -F $'\t' 'BEGIN { OFS = FS } {print $1,$2,$3,$6,$4;}' SureSelect_baits.bed > bi.txt
Figured Id share.
I had a question about the header still though, and I expect this is something I just dont understand about the conversion to bam process or something with picard.
The CalculateHSMetrics still yells at me that interval file needs a header.
It seems my aligned_reads.bam files are lacking "@HD VN:1.0 SO:coordinate" at the very top. Is this abnormal?
If I use a bam file that has gone through Picard Read group assignment it does have the @HD etc., but it also will have a @RG line as well.
So do these interval files need to be made for each sample after RG assignment?
But why not "samtools sort" added itself?
Leave a comment:
-
Ok this is really frustrating. Still is complaining I do not have header in my interval file. Here is a sample of my file...
Code:@HD VN:1.0 SO:coordinate @SQ SN:1 LN:249250621 @SQ SN:2 LN:243199373 @SQ SN:3 LN:198022430 @SQ SN:4 LN:191154276 @SQ SN:5 LN:180915260 @SQ SN:6 LN:171115067 @SQ SN:7 LN:159138663 @SQ SN:8 LN:146364022 ... @SQ SN:GL000192.1 LN:547496 @RG ID:4346_TTAGGC_ID PL:illumina PU:TTAGGC LB:4346_TTAGGC_LB SM:4346_TTAGGC_SM @PG ID:bwa PN:bwa VN:0.5.9-r16 1 45787138 45787258 + BI426105800_19859 1 45787178 45787298 + BI426105800_19860 1 45790352 45790472 + BI426105800_19867 1 45790392 45790512 + BI426105800_19868 1 45791243 45791363 + BI426105800_19875 1 45791283 45791403 + BI426105800_19876 ...
Leave a comment:
-
Originally posted by ECO View PostThat intervals file is annoying to make... here's how I do it (basically you need to add a SAM header and rearrange some columns):
Code:#Input files to CalculateHsMetrics need SAM header on an interval file ("picard interval file") #example here: ftp://ftp.broadinstitute.org/pub/gsa/exampleFiles/thousand_genomes_alpha_redesign.targets.interval_list #put header from bam file at the top of the BI file above "baits.txt" samtools view -H aligned_reads.bam > header.txt #interval file needs to look like this: #1 1104841 1104940 + target_1 #1 1105283 1105599 + target_2 #1 1105712 1105860 + target_3 #rearrange columns of baits bed file, and add SAM header awk '{print $1,$2,$3,$6,$4;}' SureSelect_baits.bed > bi.txt cat header.txt bi.txt > baits.txt
awk -F $'\t' 'BEGIN { OFS = FS } {print $1,$2,$3,$6,$4;}' SureSelect_baits.bed > bi.txt
Figured Id share.
I had a question about the header still though, and I expect this is something I just dont understand about the conversion to bam process or something with picard.
The CalculateHSMetrics still yells at me that interval file needs a header.
It seems my aligned_reads.bam files are lacking "@HD VN:1.0 SO:coordinate" at the very top. Is this abnormal?
If I use a bam file that has gone through Picard Read group assignment it does have the @HD etc., but it also will have a @RG line as well.
So do these interval files need to be made for each sample after RG assignment?
Leave a comment:
-
That intervals file is annoying to make... here's how I do it (basically you need to add a SAM header and rearrange some columns):
Code:#Input files to CalculateHsMetrics need SAM header on an interval file ("picard interval file") #example here: ftp://ftp.broadinstitute.org/pub/gsa/exampleFiles/thousand_genomes_alpha_redesign.targets.interval_list #put header from bam file at the top of the BI file above "baits.txt" samtools view -H aligned_reads.bam > header.txt #interval file needs to look like this: #1 1104841 1104940 + target_1 #1 1105283 1105599 + target_2 #1 1105712 1105860 + target_3 #rearrange columns of baits bed file, and add SAM header awk '{print $1,$2,$3,$6,$4;}' SureSelect_baits.bed > bi.txt cat header.txt bi.txt > baits.txt
Leave a comment:
-
Does anyone know what format the INTERVALS files need to be? Im using simple bed files but I keep getting this error
Exception in thread "main" java.lang.IllegalStateException: Interval list file must contain header.
I tried adding a Chr Start End header but it doesnt like this either. The simplicity of this is confusing me I guess.
Thanks.
Leave a comment:
-
Originally posted by Heisman View PostSomewhere there is a web page that has the code for each of the programs... I stumbled on it before and am not really a computer guy so don't know where to find it. But it had 250 set for that metric.
Leave a comment:
-
Somewhere there is a web page that has the code for each of the programs... I stumbled on it before and am not really a computer guy so don't know where to find it. But it had 250 set for that metric.
Leave a comment:
-
Originally posted by Heisman View PostPicard by default does +/- 250 bp. I recommend using the same file for baits and targets: you can have baits that extend past targets and hence get more coverage for baits than for target if you had all of your target sequence covered by baits. If you had say only 80% of your targets covered by baits, then you already know this, and it just complicates things to try to consider it again. So, again, I recommend using the actual bait intervals if possible.
Also, can the interval be modified (to, say, +/- 300bp)?
Leave a comment:
Latest Articles
Collapse
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...-
Channel: Articles
04-22-2024, 07:01 AM -
-
by seqadmin
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-25-2024, 11:49 AM
|
0 responses
19 views
0 likes
|
Last Post
by seqadmin
04-25-2024, 11:49 AM
|
||
Started by seqadmin, 04-24-2024, 08:47 AM
|
0 responses
17 views
0 likes
|
Last Post
by seqadmin
04-24-2024, 08:47 AM
|
||
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
62 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
60 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
Leave a comment: