Background

Background

Sequencing data analysis typically focuses on either assessing DNA or RNA. As a reminder here is the interplay between DNA, RNA, and protein:

DNA Sequencing

• Fixed copy of a gene per cell
• Analysis goal: Variant calling and interpretation

RNA Sequencing

• Copy of a transcript per cell depends on gene expression
• Analysis goal: Differential expression and interpretation

Note

Here we are working with RNA sequencing

Next Generation Sequencing

Here we will analyze a DNA sequence using next generation sequencing data. Here are the steps to get that data:

• Library Preparation: DNA is fragmented and adapters are added to these fragments

• Cluster Amplification: This library is loaded onto a flow cell, where the adapters help hybridize the fragments to the flow cell. Each fragment is then amplified to form a clonal cluster

• Sequencing: Fluorescently labelled nucleotides are added to this flow cell and each time a base in the fragment bonds a light signal is emmitted telling the sequencer which base is which in the sequence.

• Alignment & Data Analysis: These sequenced fragments, or reads, can then be aligned to a reference sequence to determine differences.

Singe End v. Paired End Data

• single-end sequence each DNA fragement from one end only
• paired-end sequence each DNA fragement from both sides. Paired-end data is useful when sequencing highly repetitive sequences.