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twang15 / K562-Analysis

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Biology 1 #6

Closed twang15 closed 2 years ago

twang15 commented 3 years ago

Transcription factors:

Operon

Operator: (also sequences of DNA)

Repressor <-> operator

RNA polymerase <-> promoter

twang15 commented 3 years ago

TSS is defined as the transcriptional start site. This is where RNA polymerase begins transcribing the DNA. This is also the beginning of the UTR (untranslated region), assuming that the gene has a 5'UTR, which is typically the case for human genes.

chip_workflow_june2017_step5

twang15 commented 3 years ago
  1. ChIPseeker can be seen as an alternative and newer workflow to ChIPpeakAnno. It also offers additional functionality, e.g. especially when it comes to visualising ChIP profiles and comparing functional annotations.
    • It supports annotating ChIP peaks
    • provides functions to visualize ChIP peaks coverage over chromosomes
    • visualize profiles of peaks binding to TSS regions
    • Comparison of ChIP peak profiles and annotation
    • supports evaluating significant overlap among ChIP-seq datasets

Coverage:

File Formats:

twang15 commented 3 years ago

DNA strand

  1. sense strand also called coding strand, plus strand, non-template strand
    • sense strand is the same as mRNA except that T in DNA is replaced by U in RNA
    • the sense strand contains codons (T in DNA is replaced by U in RNA)
    • tRNA has the anti-codon (transcribed from mRNA)
    • tRNA is used to transfer element to make an amino acid
  2. anti-sense strand also called non-coding strand, minus strand, template strand
    • anti-sense strand acts as template for the synthesis of mRNA.
    • anti-sense strand is complementary to the sense strand and mRNA (U replace T)
    • anti-sense strand contains anti-codon
    • Codon (3-bp sequence for an amino acid), anti-codon (mRNA complements this template)
  3. More details
    • DNA is double-stranded. By convention, for a reference chromosome, one whole strand is designated the "forward strand" and the other the "reverse strand". This designation is arbitrary. Sometimes the terms "plus strand" and "minus strand" are used instead.
    • Visually (I'm not talking about the transcription machinery yet), you would typically read the sequence of a strand in the 5-3 direction. For the forward strand, this means reading left-to-right, and for the reverse strand it means right-to-left.
    • A gene can live on a DNA strand in one of two orientations. The gene is said to have a coding strand (also known as its sense strand), and a template strand (also known as its antisense strand). For 50% of genes, its coding strand will correspond to the chromosome's forward strand, and for the other 50% it will correspond to the reverse strand.
    • The mRNA (and protein) sequence of a gene corresponds to the DNA sequence as read (again, visually) from the gene's coding strand. So the mRNA sequence always corresponds to the 5-3 coding sequence of a gene.
    • Now, the RNA polymerase machinery moves along the DNA in the 5-3 orientation of the coding strand (e.g. left-to-right for a forward strand gene). It reads the bases from the template strand (so it is reading in the 3-5 direction from the point-of-view of the template strand), and builds the mRNA as it goes. This means that the mRNA matches the coding sequence of the gene, not the template sequence. (This diagram from Wikipedia illustrates).
    • Annotations such as Ensembl and UCSC are concerned with the coding sequences of genes, so when they say a gene is on the forward strand, it means the gene's coding sequence is on the forward strand. To follow through again, that means that during transcription of this forward-strand gene, the gene's template sequence is read from the reverse strand, producing an mRNA that matches the sequence on the forward strand. dna
twang15 commented 3 years ago

5-prime (5') and 3-prime (3')

  1. 5-prime: A term that identifies one end of a single-stranded nucleic acid molecule. The 5' end is that end of the molecule which terminates in a 5' phosphate group. The 5' direction is the direction toward the 5' end. Nucleic acid sequences are written with the 5' end to the left and the 3' end to the right, in reference to the direction of DNA synthesis during replication (from 5' to 3'), RNA synthesis during transcription (from 5' to 3'), and the reading of mRNA sequence (from 5' to 3') during translation.
  2. 3-prime: A term that identifies one end of a single-stranded nucleic acid molecule. The 3' end is that end of the molecule which terminates in a 3' phosphate group. The 3' direction is the direction toward the 3' end. Nucleic acid sequences are written with the 5' end to the left and the 3' end to the right, in reference to the direction of DNA synthesis during replication (from 5' to 3'), RNA synthesis during transcription (from 5' to 3'), and the reading of mRNA sequence (from 5' to 3') during translation.
twang15 commented 3 years ago

Visualization Genes

  1. Built-in R package:
  2. Standalone
twang15 commented 3 years ago

ChIPseeker readPeakFile: https://rdrr.io/bioc/ChIPseeker/man/readPeakFile.html

Data structures

  1. GRanges, GrangesList: https://biodatascience.github.io/compbio/bioc/GRL.html
  2. IRanges, Normal IRanges
  3. Rle, Views, RangedData

File format

  1. bed, bigbed, narrowPeak
  2. wig, bigwig, bedGraph

Visualization

  1. covplot
    • After peak calling, we would like to know the peak locations over the whole genome, covplot function calculates the coverage of peak regions over chromosomes and generate a figure to visualize
    • Support both bed and GRangesList
    • GRangesList is also supported and can be used to compare coverage of multiple bed files.
twang15 commented 3 years ago

IRanges operations

  1. Finding overlaps: Finding (pairwise) overlaps between two IRanges
    • as(ov, "matrix") to convert an overlapping (ov) into a matrix
    • queryHits(), subjectHits() (often used with unique())
    • countOverlaps(ir1, ir2)
    • intersect
  2. Finding nearest IRanges: nearest(), precede(), follow()
twang15 commented 3 years ago

Indexing VCF

  1. One cannot create a fai from VCF file. VCF indexing produces idx files and fasta indexing generates fai files

hg19 to hg38 for SNP VCF

  1. CrossMap.py vcf hg19ToHg38.over.chain ENCFF752OAX.vcf ~/rgtdata/hg38/genome_hg38.fa ENCFF752OAX_hg38.vcf @ 2021-04-15 12:51:36: Lifting over ... @ 2021-04-15 12:53:07: Total entries: 3781183 @ 2021-04-15 12:53:07: Failed to map: 26485
  2. CrossMap.py vcf hg19ToHg38.over.chain ENCFF752OAX.vcf ~/rgtdata/hg38/genome_hg38.fa ENCFF752OAX_hg38_no_comp_allele.vcf --no-comp-allele @ 2021-04-15 13:03:24: Lifting over ... @ 2021-04-15 13:04:51: Total entries: 3781183 @ 2021-04-15 13:04:51: Failed to map: 6312

VCF to bed

  1. installation of bedops: https://github.com/bedops/bedops
  2. vcf2bed --keep-header ENCFF752OAX_hg38.bed
twang15 commented 3 years ago

Bed tools tutorial

Objective and tasks: https://github.com/twang15/K562-Analysis/issues/5

twang15 commented 3 years ago

Why intersect?

  1. By far, the most common question asked of two sets of genomic features is whether or not any of the features in the two sets “overlap” with one another.
  2. bedtools intersect works with both BED/GFF/VCF and BAM files as input.