Pilon version 1.23 introduces two new experimental arguments to specify long read input BAMs:

--nanopore ont.bam identifies ont.bam as containing long reads from Oxford Nanopore sequencing --pacbio pb.bam identifies pb.bam as containing long reads from Pacific Biosciences sequencing

In this version, the long read BAMs are only used for SNP and indel calling based on pileups. Long reads are not yet used for local reassembly or gap filling, but that will likely come in a future release. For development, I have been using minimap2 to generate the long read BAM files.

Currently, use of long reads is most effective in combination with Illumina --frags libraries, so that Pilon can use the high base quality of the Illumina libraries for unique sequence and use the long reads to reach into repeat sequence to disambiguate embedded differences. It is possible to use only long reads as input to Pilon, but consider that very experimental.

There are limitations in Pilon's use of long reads in pileups: for both --pacbio and --nanopore libraries, Pilon does not attempt to call indels in homopolymer runs of 4 or more bases (e.g., ), and for sequence, Pilon does not use the long reads to call the middle base of a motif, as the ONT base calls can be confused by methylation. So this is very basic long read support, but it has been effectively applied to more than a dozen bacterial genomes in conjunction with Illumina paired-end sequencing.