RS_Modification_and_Motif_Analysis Protocol

Use this protocol to identify common bacterial base modifications (6-mA, 4-mC, and optionally TET-converted 5-mC), and then analyze the methyltransferase recognition motifs.

• Filters reads by length and quality, maps them against a provided reference sequence, and then calls variants.

Filtering Parameters (SFilter v1)

• Minimum Subread Length: Subreads shorter than this value (in base pairs) are filtered out and excluded from analysis.
• Minimum Polymerase Read Quality: Polymerase reads with lower quality than this value are filtered out and excluded from analysis.
• Minimum Polymerase Read Length: Polymerase reads shorter than this value (in base pairs) are filtered out and excluded from analysis.

Mapping Parameters (BLASR v1)

• Maximum Divergence (%): The maximum allowed divergence of a read from the reference sequence.
• Minimum Anchor Size: The minimum size of the read (in base pairs) that must match against the reference sequence.
• Write Output as a BAM File: Specify whether or not to output a BAM representation of the cmp.h5 file.
• Write BED Coverage File: Specify whether or not to output a BED representation of the depth of coverage summary.
• Place Repeats Randomly: Specify that if BLASR maps a read to more than one location with equal probability, then it randomly selects which location it chooses as the best location. If not set, BLASR defaults to the first on the list of matches.
• Advanced Pbalign Options: Allows you to pass non-standard parameters to the underlying pbalign.py script. Use this option with care!

Consensus Parameters (Quiver v1)

• Consensus Algorithm: Specify whether to use the Plurality or Quiver consensus/variant algorithm.
• Plurality is a very simple variant-calling algorithm which does not perform any local realignment. It is heavily biased by the alignment produced by the mapper, and it is insensitive at detecting indels.
• Quiver is a more sophisticated algorithm that provides additional information about each read, allowing more accurate consensus calls. Quiver does not use the alignment provided by the mapper except for determining how to group reads together at the gross level. Quiver implicitly performs its own realignment, so it is highly sensitive to all variant types, including indels.
• Output Consensus FASTA and FASTQ Files: Specify whether or not to output FASTA and FASTQ representations of the consensus sequence.
• Write SNPs/Variants As VCF file: Specify whether or not to output a VCF representation of the variants.
• Write SNPs/Variants As BED file: Specify whether or not to output a BED representation of the variants.
• Use Only Unambiguously Mapped Reads: Specify whether or not to filter out reads where Map QV is less than 10. This reduces coverage in repeat regions that are shorter than the read length.
• Diploid Analysis: Specify whether or not Quiver operates in diploid mode. If selected, it calls variants with the assumption that there are two copies of the genome in the sample so the mapping specificity should be higher. (If not selected, Quiver operates in haploid mode.)

Postprocessing Parameters (Base Modification Detection with Motif Finding)

• Control Job ID: You can compare IPD (interpulse durations) from a control job instead of an in-silico control. If you specify a Control Job ID, no modification identification is performed as the kinetic signatures might be different.
• Identify Modifications: Specify whether or not to identify the modification type - currently m6A, m4C, and m5C with tet treatment. If you specify a Control Job ID, this option is ignored.
• Use Only Unambiguously Mapped Reads: Filter out the reads where Map QV is less than 10. This reduces coverage in repeat regions that are shorter than the read length.

Motif Finder v1

• Minimum Modification QV: The minimum quality value needed to identify a motif.