Changes between Version 10 and Version 11 of SOPs/atac_Seq

Timestamp:

08/14/20 09:58:42 (4 years ago)

Author:

gbell

Comment:

--

SOPs/atac_Seq

@@ -6 +6 @@
 
 === Basic Approach ===
- * Pre-process reads (remove adapters and other "contamination")
- * Map reads to the genome (with an unspliced mapping tool)
+ * [#preprocess Pre-process reads](remove adapters and other "contamination")
+ * [#map Map reads to the genome] (with an unspliced mapping tool)
  * Run quality control and calculate QC metrics
- * Identify ATAC-seq "peaks" with a high coverage of mapped reads
+ * [#call_peaks Call ATAC-seq "peaks"] with a high coverage of mapped reads
  * Analyze peak regions for binding motifs
  * Identify differentially accessible regions (for multiple-sample experiments)
  * Identify potential targets of peak regions and/or differentially accessible regions
 
+  * Paired-end reads are better and recommended over single reads for ATAC-seq.  The original [[https://www.nature.com/articles/nmeth.2688 | ATAC-Seq method]] from the Greenleaf lab recommends PE reads, stating, "We found that ATAC-seq paired-end reads produced detailed information about nucleosome packing and positioning."
 
-=== ATAC-Seq Analysis Details ===
+=== [=#preprocess Pre-process reads] ===
 
-  * Paired-end reads are better and recommended over single reads for ATAC-seq.  The original [[https://www.nature.com/articles/nmeth.2688 | ATAC-Seq method]] from the Greenleaf lab recommends PE reads, stating, "We found that ATAC-seq paired-end reads produced detailed information about nucleosome packing and positioning."
   * Check adapter contamination with fastqc. Remove adapters with cutadapt or trim_galore:
 
@@ -27 +27 @@
 }}}
 
-  * Map reads with bowtie2, for PE keep only concordant pairs
+=== [=#preprocess Map reads] ===
+
+  * Map reads with bowtie2, 
+  * If mapping paired-end reads, keep only concordant pairs
 
 {{{
-# --very-sensitive options --no-discordant    suppress discordant alignments for paired reads
-# -X/--maxins <int>  maximum fragment length (500). Increase to 2000 to get a better nucleosome distribution.
+# --very-sensitive 
 # --no-discordant    suppress discordant alignments for paired reads
+# -X/--maxins <int>  maximum fragment length (default=500). Increase to 2000 to get a better nucleosome distribution.
 
 bowtie2 --very-sensitive --no-discordant -p 2 -X 2000 -x /nfs/genomes/human_hg38_dec13_no_random/bowtie/hg38 -1 read1.fq -2 read2.fq | samtools view -ub - | samtools sort -T $name - >| bowtie_out.bam
 }}}
 
-  * Remove duplicates, if needed check fastqc to see deduplication level.  Remove with Picard MarkDuplicates or Samtools rmdup
-
+  * Remove duplicates with Picard MarkDuplicates or Samtools rmdup
+  * Check deduplication level with 'fastqc'
   * Remove reads mapped to mitochondria
 
@@ -45 +48 @@
 }}}
 
-  * Call peaks using MACS
+=== [=#call_peaks Call peaks] ===
 
+MACS v2 works well for this.
+
+If using the 'BAMPE' option with paired-end reads, let MACS run the pileup and calculate 'extsize'; ask MACS to keep only 1 read if duplicates are present.
 {{{
-#use this only if using BAMPE, let MACS pileup and calculate extsize; duplicates were not removed
+
 macs2 callpeak -f BAMPE -t file.sorted.bam --broad --keep-dup 1 -B -q 0.01 -g mm -n MACS_ATACSeq_Peaks
 
@@ -82 +88 @@
 
 
-==== Tips/Recommendations for ATAC-Seq ====
+=== Tips/Recommendations for ATAC-Seq ===
 //Based on presentation by H.Liu at BioC 2020 presentation //
   *  post-alignment filtering: remove mito/ChrM reads, remove duplciates, remove mapping artifacts: < 38bp and fragments > 2kb, and discordant reads