Biology-2

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Splicing mechanism Intron

• starting point: 5' (5 prime) splice site: splice donor, GT***
• ending point: 3' (3 prime) splice site: splice acceptor, ***AG
• branch site: A
• intron are much longer, a few hundreds bps up to tens of thousands of hundreds of bps exon are 25~300 bp
• starting site: **AG (intron's ending site)
• ending site: GT** (intron's starting site)
• exon are like islands in the Pacific Ocean of introns

Intron facilitate evolution of new genes Alternative splicing greatly increases proteome complexity Higher eukaryotes utilize exon definition to define splice site

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A, T, C, G, N

We know that the four native bases for DNA are AGTC, however, some of the sequences, retrieved from NCBI, contain letter 'N', which illustrates that these nucleotide bases are not deciphered correctly, leaving an unidentified nucleotide.

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Chimeric reads occur when one sequencing read aligns to two distinct portions of the genome with little or no overlap. Chimeric reads are indicative of structural variation. Chimeric reads are also called split reads.

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Illumina Sequencing by Synthesis

1. Library Prep: oligo
2. Clustering: PCR amplification
3. Sequencing
   • forward read
   • reverse read
   • To have read 1 and read 2 in the same orientation, reverse-complement one of the two reads.
   • Keep in mind though, you usually do not know, which of the two reads corresponds to the forward strand of your initial template.
   • Illumina paired-end sequencing is based on the idea that you have initial DNA fragments (longer than your actual read length) and you sequence both its ends. On the Illumina chip, both ends of each sequence are amplified prior to actual sequencing using bridging. This approach results in two reads per fragment, with the first read in forward orientation and the second read in reverse-complement orientation. Depending on the inital fragment size and read length, these fragment can either overlap or not
4. Data analysis
   • transfer
   • store
   • align
   • analysis
   • etc

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mRNA post-transcription modification

1. RNA-edit: call A to G change if the gene is on plus strand; T to C change if the gene is on minus strand
   • in this case you're going to want to use pysam, since I assume you have strand-specific RNAseq data and you'll need to process reads according to the strand from which each fragment originated, rather than how the read itself aligned.
   • For double-stranded genomic DNA there is transcribed stuff on both strands. In standard protocols the generation of fragments is random, as is the strand they originate from.
   • For RNAs, there obviously is a forward strand. But only in case of strand-specific RNA-seq you actually know how your reads are oriented with respect to your inital template.

DNA replication

1. DNA helicases
   • There are DNA and RNA helicases. DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied.
2. Primase: enzyme to make a small piece of RNA called primer. The primer marks the starting point of the construction of the new strand of DNA
3. DNA polymerase: enzyme to bind to the primer and make the new strand. It can only add DNA base from 5' to 3' direction. (From perspective of template strand, DNA polymerase moves from 3' to 5')
   • the leading strand is made continuously
   • the lagging strand cannot be made continuously. The DNA polymerase can only make this strand in small chunks, called Okazaki fragment.
   • • each fragment starts with a RNA primer
   • • then DNA polymerase adds small chunk of DNA bases from 5' to 3' direction
   • • the next primer is added further down the lagging strand
   • • repeat the above two steps until a whole strand is finished.
   • • exonuclease (an enzyme) removes all RNA primers from both strand of DNA and another DNA polymerase fills the gap left behind (occupied by RNA primers) with DNA bases
4. DNA ligase (an enzyme) seals up the fragments of DNA for both new replicated strands to form continous double strand.

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Stranded vs. Non-stranded RNA

1. single-read vs. paired-read
2. DNA vs RNA
3. can a read Map to both forward strand and reverse strand at the same time?
4. which strand, forward or reverse, is the reference genome?
5. cDNA: complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase.

Differential RNA processing

1. nRNA is pre-mRNA

Galaxy: read mapping

1. https://training.galaxyproject.org/training-material/topics/sequence-analysis/tutorials/mapping/tutorial.html

RNA-seq

1. reveal the presence and quantity of RNA in a biological sample at a specific development stage or physiological condition
   • interpret the functional elements of the genome
   • understand development and disease
   • reveal molecular constituents of cells and tissues
2. Third generation sequencing technology (such as PacBio) enable PCR-free (avoid PCR bias) and full-length transcripts sequencing

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Primary transcript, mRNA, nRNA, tRNA, hnRNA, pre-RNA

Three types of Primary transcript: A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs.

• pre-mRNA (nRNA or precursor RNA): Pre-mRNA is synthesized from a DNA template in the cell nucleus by transcription, and then undergoes RNA splicing/processing/editing to mRNA (mature messenger RNA or messenger RNA). The initial transcript from a protein-coding gene is often called a pre-mRNA
• hnRNA: Pre-mRNA comprises the bulk of heterogeneous nuclear RNA (hnRNA). The term hnRNA is often used as a synonym for pre-mRNA, although, in the strict sense, hnRNA may include nuclear RNA transcripts that do not end up as cytoplasmic mRNA.