Changes between Version 38 and Version 39 of SOPs/atac_Seq

Timestamp:

03/16/21 13:35:02 (4 years ago)

Author:

thiruvil

Comment:

--

SOPs/atac_Seq

@@ -136 +136 @@
 Additional notes on calling peaks using MACS: ''using MACS default options without BAMPE will not call the correct peaks''.  Alternative options/parameters in calling peaks using MACS, if BAMPE is not used are, --nomodel --shift s --extsize 2s, this can be used for single-end reads as well, e.g. --shift 100 --extsize 200 is suitable for ATAC-Seq.   
     * MACS' author, T.Liu, recommends using -f BAMPE if PE reads are used [[https://github.com/taoliu/MACS/issues/331]], using BAMPE option asks MACS to pileup and calculate the extension size -  works for finding accessible regions within cut sites.  The additional parameters can also be used to look only at the //exact// cut sites by Tn5 instead of the open/accessible regions [[https://github.com/taoliu/MACS/issues/145]], if so, -f BAMPE may not be suitable.
-      * Shifting reads, pos. strand + 4 and neg strand -5 (see recommendations below) may be needed as well to find //exact// cut sites.
+      * Shifting reads, pos. strand +4 and neg strand -5 (see recommendations below) may be needed as well to find //exact// cut sites.
     * another approach is to convert the bam file to bed (using bedtools), and use the options, -f BED --shift 100 --extsize 200
 
@@ -243 +243 @@
   * For calling open/accessible regions, at least ~50M reads are recommended.
   * For transcription factor footprinting, at least ~200M reads are recommended to ensure high coverage of the NFR.
-  * 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: NFR, mono, di, etc. Shifted reads that do not fit into any of the bins should be discarded.
+  * 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: NFR, mono, di, etc. Shifted reads that do not fit into any of the bins should be discarded.
   * Use housekeeping genes to check QC: signal enrichment is expected in the regulatory regions of housekeeping genes in good ATAC-seq experiments. 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.