Problem Picard "Alignment start should != 0 because reference name != *"

[MurielGB]

Hello,

Using Picard Validation, I obtain this message for some reads : Alignment start should != 0 because reference name != *
If I grep the read in the SAM file, I can see that the read is mapped (there is the name of a reference scaffold in the third column) but the position (column 4) is 0.
This is very strange, how can this happen ?
Do you know how to fix this ?

Thanks,

Muriel

[dpryan]

What's the flag on that read?

[MurielGB]

Hello,

After doing bwa aln and bwa sampe, I obtain a sam file and I use Picard ValidateSamFile.
The errors are :

ERROR: Record 38208, Read name HWI-ST1206:14:C296WACXX:6:1101:8672:9595, Mate negative strand flag does not match read negative strand flag of mate
ERROR: Record 40751, Read name HWI-ST1206:14:C296WACXX:6:1101:8820:10033, Alignment start should != 0 because reference name != *.

If I grep the read with the second problem, here is what I obtain :

HWI-ST1206:14:C296WACXX:6:1101:8820:10033 113 gi|300388125|ref|NC_013991.2| 0 25 46M =176 215 GTACTTTTTTTTTTAGTATTTTAATTTTGTATTTATTATTTATATT >GCGAGGEIIIHHGHHIHE@HHC:CA<<AAGC>DFHFFDDDDD@@<RG:Z:Pa1370-Seq1 XT:A:U NM:i:0 SM:i:25 AM:i:25 X0:i:1 X1:i:1 XM:i:3 XO:i:0 XG:i:0 MD:Z:0 XA:Z:gi|300388125|ref|NC_013991.2|,-86195,4M1D42M,1;

HWI-ST1206:14:C296WACXX:6:1101:8820:10033 177 gi|300388125|ref|NC_013991.2| 176 37 85M =0 -215 GCAGCTAGGTCTAGAGGGAAGTTGTGAGCATTACGTTCATGCATTACTTCCATACCAAGGTTAGCACGGTTGATGATACCAGCCC @@@@@>@@>;;;AA=>BBBA<BBBBAA:BA=;B=9??ABBBBBBBBAA>BA<?*BABBB>C?AC7<CC@>A>72BC@@)=<A=== RG:Z:Pa1370-Seq1 XT:A:UNM:i:1 SM:i:37 AM:i:25 X0:i:1 X1:i:0 XM:i:1 XO:i:0 XG:i:0 MD:Z:78T6

Any idea on how I can have a read map but no position given ???

Thanks !

Muriel

[dpryan]

That would seem to be a bug.

[MurielGB]

OK thanks !
Any idea on what I can do ?

[dpryan]

Try emailing the list.

[MurielGB]

OK, i will, thanks for your help !
Muriel

[glopez]

Hi Muriel,

I am facing the same problem. Did you find a solution for it?

Thanks and kind regards

[MurielGB]

Hello !
Well, what I did is a personal arrangement. I should probably write to the mailing list, I don't remember if I did or not ahah !!
The problem is that some read are supposedly mapped on the reference because there is a scaffold name in the column 3, but the starting position (column 4) of the read is 0 and this is the problem.
Here is what I did :

### List reads that have "Alignment start should != 0 because reference name != *"
samtools view -h input.bam | awk 'BEGIN {OFS="\t";} {if ($1=="^@") {print $0;} else {if ($4==0 && $3!="*") {$4=1; print $1>"read_file"} if ($8==0 && $7!="*") {$8=1;print $1>"read_file"} print $0;}}' | samtools view -bS - > input_OK.bam ; sort read_file | uniq > read_file_sorted

### Exclude problematic reads
#java -Xmx20g -jar FilterSamReads.jar INPUT=INPUT_OK.bam FILTER=excludeReadList READ_LIST_FILE=read_file_sorted OUTPUT=output.bam

Good luck !

Muriel

[glopez]

Hi Muriel,

THanks for your reply!!

Kind regards