CRISPResso2 | deep sequencing data for rapid and intuitive interpretation | Genomics library

Please check that your input file(s) are in FASTQ format (compressed fastq.gz also accepted). If you get an empty report, please double check that your amplicon sequence is correct and in the correct orientation. It can be helpful to inspect the first few lines of your FASTQ file - the start of the amplicon sequence should match the start of your sequences. If not, check to see if the files are trimmed (see point below). It is important to determine whether your reads are trimmed or not. CRISPResso2 assumes that the reads ARE ALREADY TRIMMED! If reads are not already trimmed, select the adapters used for trimming under the ‘Trimming Adapter’ heading under the ‘Optional Parameters’. This is FUNDAMENTAL to CRISPResso analysis. Failure to trim adaptors may result in false positives. This will result in a report where you will observe an unrealistic 100% modified alleles and a sharp peak at the edges of the reference amplicon in figure 4. The quality filter assumes that your reads uses the Phred33 scale, and it should be adjusted for each user’s specific application. A reasonable value for this parameter is 30. If your amplicon sequence is longer than your sequenced read length, the R1 and R2 reads should overlap by at least 10bp. For example, if you sequence using 150bp reads, the maximum amplicon length should be 290 bp. Especially in repetitive regions, multiple alignments may have the best score. If you want to investigate alternate best-scoring alignments, you can view all alignments using this tool: http://rna.informatik.uni-freiburg.de/Teaching/index.jsp?toolName=Gotoh. As input, sequences from the 'Alleles_frequency_table.txt' can be used. Specifically, for a given row, the value in the 'Aligned_Sequence' should be entered into the 'Sequence a' box after removing any dashes, and the value in the 'Reference_Sequence' should be entered into the 'Sequence b' box after removing any dashes. The alternate alignments can be selected in the 'Results' panel in the Output section.