MARGI is a protocol for mapping RNA-DNA contacts on a genome-wide scale, analogous to Hi-C methods which map DNA-DNA contacts. In situ MARGI (iMARGI) is the successor of the MARGI technique, requiring fewer input cells and less time than required by MARGI.
\nThe 4DN iMARGI data processing pipeline is adapted from the Zhong iMARGI pipeline. Its primary components are cleaning and alignment of reads, parsing of alignments into pairs, and merging and aggregation of pairs. To learn more about the original pipeline or experimental protocol, please reference the iMARGI Pipeline documentation.
\nThe primary modifications are:
\ncooler cload pairs, andThe iMARGI Docker, used in all steps of the pipeline, can be found at https://hub.docker.com/r/4dndcic/imargi/v1.1.1_dcic_4
In iMARGI experiments, two random bases initiate each RNA end read. Thus to improve mapping, R1 reads are cleaned using seqtk version 1.3. The command
\nseqtk trimfq -b 2\n
removes two bases () from the left end of each read.
Reads are then mapped to the GRCh38 (human) or mm10 (mouse) reference genome using bwa version 0.7.17. In particular, we run:
\nbwa mem -t <nthreads> -SP5M <genome_index> <fastq1> <fastq2>\n
-SP option is used to ensure the results are equivalent to that obtained by running bwa mem on each mate separately, while retaining the right formatting for paired-end reads. This option skips a step in bwa mem that forces alignment of a poorly aligned read given an alignment of its mate with the assumption that the two mates are part of a single genomic segment.-5 option is used to report the 5' portion of chimeric alignments as the primary alignment. For chimeric alignments, bwa mem reports two alignments: one of them is annotated as primary and soft-clipped, retaining the full-length of the original sequence. The other end is annotated as hard-clipped and marked as either 'supplementary' or 'secondary'.-M option is used to annotate the secondary/supplementary clipped reads as secondary rather than supplementary, for compatibility with some public software tools such as picard MarkDuplicates.-t option is used for multi-threading and should not affect the result.Source files (v1.1.1_dcic_4):
Interaction pairs are parsed from the bam files using pairtools version 0.2.2. Filtering consists of several commands:
pairtools parsebam file.For more details, see the pairtools documentation.
\npairtools sort
pairsam file from an input pairsam file.Note that the flipping order and sort order of chromosomes is not identical. See the docs for more details.
\npairtools dedup --mark-dups
pairtools markasdup)Arbitrarily designate the duplicate status among the two duplicate alignments.
\npairtools select
Source files (v1.1.1_dcic_4):
\nPairs are merged and aggregated with pairix version 0.3.3. Pairs files are then converted to mcool via cooler version 0.8.5.
Merging:
\npairs files using run-merge-pairs.sh.pairs file.File Format Conversion:
\nmcool files are contact matrices containing multiple resolutions which can be visualized in HiGlass.mcool files are: 1kb, 2kb, 5kb, 10kb, 25kb, 50kb, 100kb, 250kb, 500kb, 1Mb, 2.5Mb, 5Mb, 10Mb.Source files (v1.1.1_dcic_4):
\nThe 4DN version of the iMARGI pipeline also contains an output QC report and summary statistics generated on output pairs files. See an example report here.
Source files (v1.1.1_dcic_4)
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