Hi Yibo,
It would help if you send the stack trace that should be printed along
with the error. However, the fact that you need to reduce the
validation stringency on the various PIcard tools means that you are
likely to run into various problems. I suggest you change your workflow
as follows: rather than using the output of your aligner directly, use
Picard MergeBamAlignment to combine the aligner output with a valid
unmapped BAM. This should eliminate all the Picard validation issues.
If you don't have an unmapped BAM, you can create one with Picard
FastqToSam.
-Alec
On 7/16/14, 3:18 PM, 胡义波 wrote:
Dear all,
I am using Picard tools but encountered some problems. I want to use GATK to
call snp for RNA-seq data. I have used two methods, Tophat2 and STAR, to
perform RNA-seq mapping. Because I do not have my own reference genome, I used
the genome of a related species as the reference (different genus). However,
for the Tophat2 and STAR mapping output (I used only unique mapping output),
when using Picardtools (SortSam, AddOrReplaceReadGroups or MarkDuplicates),
some errors occurred.
I am using Java 1.7.0_45 and Picard-tools-1.117 (or 1.77). The error messages
are as follows:
(1) For STAR output, I successfully used AddOrReplaceReadGroups to add read
groups and sort by coordinate. However, when using MarkDuplicates, an error
occurred as follows (running log attached):
"Exception in thread "main" htsjdk.samtools.SAMException: Exception when processing
alignment for BAM index HS2:410:C21E6ACXX:4:1109:16160:63599 2/2 99b aligned read."
I checked this read and it is a read pair and seems ok. I attached the
paired-end reads information as follows:
HS2:410:C21E6ACXX:4:1109:16160:63599 163 1 536940431 255
9S90M = 536940464 99
CAGAAAATGAACTGTTTGGAGAAAGATGCCTGCAGAAACCTACATAGCAGCAAGAAGATCCAGAATGAATTTTGGGGTACAATTGATTGAACTGAAGGG
BBBFFFFFFFFFFIFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFF<BBFFFFFFFFFBFFFFFFFFF
RG:Z:LANE11A NH:i:1 HI:i:1 jI:B:i,-1 jM:B:c,-1 nM:i:10 AS:i:134
HS2:410:C21E6ACXX:4:1109:16160:63599 83 1 536940464 255
66M33S = 536940431 -99
CATAGCAGCAAGAAGATCCAGAATGAATTTTGGGGTACAATTGATTGAACTGAAGGGGGCTGAACACAATTATTTTGAATGTAAACTCTTATGCCAAAG
FFFFFFFBFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFIIIIIIIIIIIIIIIIIIFIFFFFFFFFFFBBB
RG:Z:LANE11A NH:i:1 HI:i:1 jI:B:i,-1 jM:B:c,-1 nM:i:10
AS:i:134
Additionally, I used ValidateSamFile tool to check my sam file, and found as
follows:
Error Type Count
ERROR:MATE_NOT_FOUND 37
WARNING:MISSING_TAG_NM 48684343
Because only 37 reads is mate_not_found, I deleted these reads from my sam
file. Then rerunning MarkDuplicates, the same error still occurred.
My scripts are as follows:
java -Xmx4G -jar /u/home/y/ybhu/picard-tools-1.117/AddOrReplaceReadGroups.jar
INPUT=${path_input}/${samp}_uniquelymapped.sam
OUTPUT=${path_input}/${samp}_rg.sam SORT_ORDER=coordinate RGID=LANE11A
RGPL=ILLUMINA RGLB=LIB1A RGPU=LANE1 RGSM=1A
java -Xmx4G -jar /u/home/y/ybhu/picard-tools-1.117/MarkDuplicates.jar
I=${path_input}/${samp}_rg.bam O=${path_input}/${samp}_dedup.bam
METRICS_FILE=${path_input}/${samp}_dedupmetrics.txt REMOVE_DUPLICATES=TRUE
CREATE_INDEX=TRUE ASSUME_SORTED=TRUE VALIDATION_STRINGENCY=SILENT
(2) For Tophat2 output, I used different steps (first use SortSam to sort by
coordinate, then AddOrReplaceReadGroups to add read groups). However, for these
two steps, error messages also occurred (running log as attached).
For SortSam tool, 11449 reads were reported "Insert size out of range". Then I changed the argument
"VALIDATION_STRINGENCY=STRICT" to "LENIENT", the SortSam can ignore these errors and
finish sorting.
But when running AddOrReplaceReadGroups, a same error occurred (similar to STAR
running error):
"Exception in thread "main" net.sf.samtools.SAMException: Exception when processing
alignment for BAM index HS2:410:C21E6ACXX:4:2316:13634:18930 1/2 97b aligned read."
I pasted the read information here:
HS2:410:C21E6ACXX:4:2316:13634:18930 73 1 536905899 50
32M2D57M4I4M * 0 0
GTAAATTTTTGTAAATGTTCAATGAGGTGCTGAAACATGTATGTTCTTTAGCTTTCCCATTTAGAAGACAACATAAATCTTAATTTTTCCACTAATT
B<BFFFFFFFFFFFF0<BFFIIIF0BFBFFFIIIIIBFIBFFFBFFFIFFBFFFFFFFFFFIIBFFIIIFIIIIIFIFFFFFFFFBFFFBBBFFFBF
AS:i:-126 XN:i:0 XM:i:20 XO:i:2 XG:i:6 NM:i:26
MD:Z:2C1G10A3T0G2A0T0A3A0A1^AA3T9G4A1A0G2T24T1A0A5G2 YT:Z:UU NH:i:1.
My scripts are as follows:
java -Xmx3G -jar /u/local/apps/picard-tools/1.77/SortSam.jar
INPUT=${path_input}/${samp}_uniquelymapped.sam
OUTPUT=${path_input}/${samp}_unique_sorted.sam SORT_ORDER=coordinate
CREATE_INDEX=TRUE VALIDATION_STRINGENCY=LENIENT
java -Xmx3G -jar /u/local/apps/picard-tools/1.77/AddOrReplaceReadGroups.jar
INPUT=${path_input}/${samp}_unique_sorted.bam
OUTPUT=${path_input}/${samp}_rg.bam SORT_ORDER=coordinate CREATE_INDEX=TRUE
RGID=LANE11A RGPL=ILLUMINA RGLB=LIB1A RGPU=LANE1 RGSM=1A
VALIDATION_STRINGENCY=LENIENT
In summary, the errors for STAR and Tophat2 seem to be caused by creating BAM
index for these reported reads. But I do no know why and how to resolve it.
Your comments are very appreciated, and thanks in advance.
Yibo
Department of Ecology and Evolutionary Biology,
University of California, Los Angeles
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