Dear All,

I wonder if any of you could help me solving this puzzle: I used STAR for aligning a paired-end read: the command was :

STAR --runMode alignReads --genomeDir IndexRef/GRCg6a/ --outSAMtype BAM SortedByCoordinate --readFilesIn R0629-S0002_L10AU2_A56593_1_HGFCJDSX2_TCGTCTGA-TCAAGGAC_L003_R1_trimmed.fastq R0629-S0002_L10AU2_A56593_1_HGFCJDSX2_TCGTCTGA-TCAAGGAC_L003_R2_trimmed.fastq --outFileNamePrefix mapped/L10/BAM_L10_GRC6a/L10A2 --runThreadN 16

I did not use the option (--bamRemoveDuplicatesType). The BAM file I obtained did not contain any duplicates as revelaed by using command :

samtools view -c -f 1024 file.BAM 

It was surprising since I expected presence of some duplicates reads, and then I relaized that they might be hidden in the BAM file and thus not readable (Am I right in this ? ), so I went on to use fixmates, after sorting by names, using these command:

1. sort the BAM file by name (samtools sort -n L10AAligned.sortedByCoord.out.bam -o L10A_sortedbynames.bam)
2. apply fixmat, mc: samtools fixmate -m L10A_sortedbynames.bam filefixmate.bam
3. sort it again by coordinates (samtools sort filefixmate.bam -o filefixmatesortedbycordinates.bam)

On the resultant file, I checked the duplicates, it was also 0. I also doubled check it using flagstats, was 0

I am surprised how can I make sure that PCR duplicates are removed after fixmate them? It is also possible that the first BAM file produced from STAR is by default contains no duplicates?

Any comment?

Thanks