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  • Pengfei Liu
    replied
    Originally posted by blubucki View Post
    Hello everyone, this is my 1st post on this great forum and I look forward to your help.
    I am trying to find the chimeric/fusion/split reads in my WGS NGS library. I tried bwasw with SE reads or PE alignment,.but not sure how it flags the chimeric/split reads in the output sam file. Or if there's another way to find them.
    Can someone please help me in this.
    Thanks a lot.
    Best!
    Sorry, I use bwa-mem to do alignment. I have tested, in that, -a , -M will affect the output. -a will produce all alignment (so, secondary alignment with 0x100 will appear), and chimeric alignment will with 0x800. but if you use both -a and -M, chimeric alignment and secondary alignment will all convert to 0x100. with -M but not -a, 0x100 is chimeric alignment.
    see also"With 0.7.5 if read has a chimeric alignment, the paired or the top hit uses soft clipping and is marked with neither 0x800 nor 0x100 bits, All the other hits part of the chimeric alignment will use hard clipping and be marked with 0x800 if option "-M" is not in use, or marked with 0x100 otherwise." (https://github.com/lh3/bwa/blob/master/NEWS; );Other hits part of a chimeric alignment are now reported in the SA tag, conforming to the SAM spec v1.5"

    For Bwa-sw, in the manual it says that:'in the paired end mode, bwasw may still output split alignments but they are all marked as not properly paired;' so at least it is in the -F 2 filed, for chimeric alignment, maybe -f 0x100 or 0x800, for fusion, sorry, I do not know what does it mean.

    you may also post your question on biostar or the mailing list of bwa on sourcefore.

    Leave a comment:


  • blubucki
    replied
    Hello everyone, this is my 1st post on this great forum and I look forward to your help.
    I am trying to find the chimeric/fusion/split reads in my WGS NGS library. I tried bwasw with SE reads or PE alignment,.but not sure how it flags the chimeric/split reads in the output sam file. Or if there's another way to find them.
    Can someone please help me in this.
    Thanks a lot.
    Best!

    Leave a comment:


  • Pengfei Liu
    replied
    2 In the bwa-mem document, what does unpaired paired-end reads mean(-a parameter description)? what is the differences between it and singleton?

    3, does the chimeric alignment get a mapping quality 0?

    Leave a comment:


  • Pengfei Liu
    replied
    Thanks
    so still some additional questions
    1, I want to use the mapping results of bwa to do Differential expression analysis, what kind of reads should be kept?
    1) mapping quality q>threshold(30 or 60), as you mentioned quality larger than 1 will exclude reads mapping to multiple positions
    2) NM tag indicates the number of mismatch, so NM:i:0-5 might be used to restrict mismatch. ()
    3) flag 99-147 or 83-163 to kept properly paired reads
    are the above three suitable criteria?
    Thank you!

    Leave a comment:


  • lh3
    replied
    Hard to say. It uses seed-and-extend. As long as mismatches/gaps do not reduce the alignment score, they are allowed. When aligning fosmids, bwa-mem may allow thousands of mismatches and gaps.

    Leave a comment:


  • gmarco
    replied
    I'm also interested on this information.

    Leave a comment:


  • how many mismatches does BWA-MEM tolerate during mapping

    Hi,
    Recently I am trying to use BWA-MEM to do alignment on PE-90 reads, I have some questiones list as following;


    1,how many mismatches does BWA-MEM tolerate during mapping?
    Does the following still applied to BWA-MEM:
    ‘Given a read of length m, BWA only tolerates a hit with at most k differences
    (mismatches or gaps), where k is chosen such that <4% of m-long reads with
    2% uniform base error rate may contain differences more thank. With this
    configuration, for 15–37 bp reads,kequals 2; for 38–63 bp,k=3; for 64–92
    bp,k=4; for 93–123 bp,k=5; and for 124–156 bp reads,k=6.’(Li and Durbin, 2009)

    2,In bwa-aln sampe,the mismatch number could be setted by the ‘-n’option。
    By default,what is the number of mismatch setted and how to set it in BWA-MEM?

    3, Mapping quality is assigned for individual read, not for a read pair.so if I use '-q 30' to extract PE reads, some of the reads will lose their mate-pair, am I right? Are there any ideas on how to extract pair-reads (reads and its mate) with '-q 30' for both of them?

    I have read the manual and the BWA-MEM paper, but sitll didn't get answers.

    Any suggestions are appreciated!

    Pengfei Liu

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