AbstractSummaryThe FASTQ+ format is designed for single-cell experiments. It extends various optional tags, including cell barcodes and unique molecular identifiers, to the sequence identifier and is fully compatible with the FASTQ format. In addition, PISA implements various utilities for processing sequences in the FASTQ format and alignments in the SAM/BAM/CRAM format from single-cell experiments, such as converting FASTQ format to FASTQ+, annotating alignments, PCR deduplication, feature counting and barcodes correction. The software is open-source and written in C language.Availability and implementationhttps://doi.org/10.5281/zenodo.7007056 or https://github.com/shiquan/PISASupplementary informationSupplementary data are available at Bioinformatics online.