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  • UCSC Encode files issue with MAPQ and Flags

    Hi,

    I have been reading Seqanswers for quite some time, but this my first question.

    Does someone has experience with BAM files from UCSC-encode project? I am interested in a small RNASeq dataset from CSHL and I found some issues in the BAM files hosted at UCSC.

    It seems there are multiple alignments (2) for the same read, but not flagged as primary and secondary. In the first example bellow, same read name appears twice with same flag (0) and same alignment score (254). In the second example the duplicated read name is the reverse complement sequence.

    Code:
    # Number of multiple alignments in a sample
    samtools view ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlShortRnaSeq//wgEncodeCshlShortRnaSeqA549CellShorttotalTapAlnRep1.bam | head -n 10000| awk '{print $1}' | sort | uniq -c | awk '{print $1}' | sort | uniq -c
       9958 1
         21 2
    
    # Example 1
    view ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlShortRnaSeq/wgEncodeCshlShortRnaSeqA549CellShorttotalTapAlnRep1.bam | grep -m 2 'TUPAC_0038:7:110:15042:19226#0/1'
    TUPAC_0038:7:110:15042:19226#0/1        0       chr1    45243516        254     34M2S   *       0       0       CTCGTGATGAAAACTTTGTCCAGTTCTGCTACTGAA Ycacc\KW_RSLWSVMTTYT]a\a_`_KXK\Z\RQ_
    TUPAC_0038:7:110:15042:19226#0/1        0       chr1    45244067        254     3S31M2S *       0       0       CTCGTGATGAAAACTTTGTCCAGTTCTGCTACTGAA Ycacc\KW_RSLWSVMTTYT]a\a_`_KXK\Z\RQ_
    
    # Example 2
    samtools view ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlShortRnaSeq/wgEncodeCshlShortRnaSeqA549CellShorttotalTapAlnRep1.bam | grep -m 2 'TUPAC_0038:7:88:9054:14403#0/1'
    TUPAC_0038:7:88:9054:14403#0/1  16      chr1    234792  246     18S18M  *       0       0       CCGATCTTTTTTTTTTTCTAAGGACATCCTAAAGGA    ghfeecchhhhfhhhhhhghhhhhhhhhhhhhhhhh
    TUPAC_0038:7:88:9054:14403#0/1  0       chr1    464897  246     18M18S  *       0       0       TCCTTTAGGATGTCCTTAGAAAAAAAAAAAGATCGG    hhhhhhhhhhhhhhhhhghhhhhhfhhhhcceefhg

    Related to it, the alignment scores are also different from what I have normally seen in BAM files. It ranges from 246 to 255:

    Code:
    samtools view ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlShortRnaSeq/wgEncodeCshlShortRnaSeqA549CellShorttotalTapAlnRep1.bam | head -n 10000 | awk '{print $5}' | sort | uniq -c
         13 246
          5 247
        149 248
         11 249
         19 250
         26 251
         93 252
       9587 253
         77 254
         20 255

    Is this normal? Am I missing something? It seems to happen only for a small percentage of reads. Would you just ignore those with multiple alignments for further analysis? If so, how would you filter them out?

    Thanks!!

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