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MRNM stands for "Mate reference index". So Picard found something in the RNEXT field which should be set only for paired-end reads but the rest of the file looks like single-end. The problematic line in your code is: line.next_reference_id = 0 This sets the RNEXT SAM field to whatever Pysam stores as a reference with index 0 (next_reference_id). ...

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