Pacbio smrt analysis platform

The PacBio SMRT analysis platform (version 2.3.0) was used to detect DNA 6mA modification¹. The analysis pipeline was as follows: First, the raw SMRT sequencing datasets in h5 format downloaded from NCBI were filtered by using filter_plsh5.py with parameters: “-seed = 1 -minAccuracy = 0.75 -minLength = 50,” and the reads containing adapters, short reads (less than 50 nucleotide) or reads with a low quality region (less than 0.75) were removed. Second, the clean reads were aligned to the corresponding reference genome using pbalign with the parameters “-algorithmOptions = ‘-useQuality’ -algorithmOptions = ‘-minMatch 12 -bestn 10 -minPctIdentity 70.0’.” Then, the polymerase kinetics data were loaded by loadChemistry.py and loadPulses scripts with “-metrics DeletionQV, IPD, InsertionQV, PulseWidth, QualityValue, MergeQV, SubstitutionQV, DeletionTag.” Finally, the aligned datasets were sorted using cmph5tools, and 6mA sites were detected using ipdSummary.py script with “-methylFraction -identify 6mA -numWorkers 4.” Then we retained 6mA sites with more than 25-fold coverage for further analysis.

The genomic SMRT sequencing reads raw data of P. vaginatum were acquired by our research group in the early stages but haven’t been officially published. N6-methyladenine DNA modifications of P. vaginatum genome were detected by the PacBio SMRT analysis platform (version 2.3.0)¹ (Xiao et al., 2017 (link)). The detailed analysis workflows were as follows: the raw reads were initially aligned to the corresponding reference genome using pbalign with the parameters -seed = 1-minAccuracy = 0.75 – minLength = 50 –concordant – algorithmOptions = “- useQuality” – algorithmOptions = “- minMatch 12 -bestn 10 - minPctIdentity 70.0.” The polymerase kinetics information was loaded following alignment by loadChemistry.py and loadPulses scripts with parameters “-metrics DeletionQV, IPD, InsertionQV, PulseWidth, QualityValue, MergeQV, SubstitutionQV, DeletionTag.” Then cmph5tools was used to sort the post-aligned datasets. Finally, 6mA modification was identified by using ipdSummary.py script with parameters “- methylFraction - identify 6mA - numWorkers 4”; 6mA sites with coverage of no less than 25× were screened out for further analysis.