For paired-end sequencing, two FASTQ files (R1 and R2, or read 1 and 2) are generated in pair for input into CUTANATMCloud. This is because paired-end sequencing reads the template in two directions (forward and reverse), corresponding to the i5 and i7 indices added during library preparation. Reading in both directions enhances the fidelity of flow cell clustering and subsequent base calling, which leads to high-quality alignments. For alignment and upload to CUTANATM Cloud, both R1 and R2 FASTQ files from the same CUT&RUN reaction are required with the same file name except for the R1 and R2 designations, and will be concatenated during the alignment process.
To ensure compatibility with the Reaction Sheet for CUTANATM Cloud analysis, FASTQ file names must start with an alphanumeric character (A-Z, 0-9). Depending on how and where these files are generated, some renaming may be necessary. EpiCypher suggests 2x50bp sequencing for CUT&RUN libraries, though deviations from this standard may be unavoidable due to core facility requirements or instrument settings.