| | 84 | ==== Tips/Recommendations for ATAC-Seq ==== |
| | 85 | //Based on presentation by H.Liu at BioC 2020 presentation // |
| | 86 | * post-alignment filtering: remove mito/ChrM reads, remove duplciates, remove mapping artifacts: < 38bp and fragments > 2kb, and discordant reads |
| | 87 | * The NRF should be > 38bp to < 147 bases (one nucleosome); mono nucleosome fragments are in the range of 147 -200 bp |
| | 88 | * don't recommend to remove di-, tri nucleosome fragments for downstream analysis unless you have a great number of reads. Otherwise the you might miss many open chromatin regions during peak calling. |
| | 89 | * check fragment size dist (from ATACSeqQC) |
| | 90 | * Tn5 produces 5’ overhangs of 9 bases long: pos. strand + 4 and neg strand -5 (shiftGAlignmentsList and shiftReads function) splitGcAlignmentsByCut: creates different bins of reads: nucleosome free regions (NFR), mono, di, etc. Shifted reads that do not fit into any of the bins should be discarded. |
| | 91 | * use housekeeping genes to check QC: signal enrichment is expected in the regulatory regions of housekeeping genes in good ATACSeq eperiments. Use IGVSnapshot function with geneNames param. splitGAlignmentsByCut: creates different bins of reads: NFR, mono, di, etc. Shifted reads that do not fit into any of the bins should be discarded. |
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