5.2.1 Quantification

RNA-seq and nascent RNA-seq are two techniques used to investigate gene expression, but they differ in the type of RNA that is analyzed. Nascent RNA-seq is concerned with the examination of newly synthesized RNA molecules that are still connected to the template DNA strands from which they are being transcribed. Nascent RNA-seq quantification is a technique utilized to assess the quantity of newly synthesized RNA in a sample. It investigates nascent RNA molecules that are still connected to the template DNA strands from which they are being transcribed.

5.2.2 What it does?

The quantification module is a wrapper script to deal with nascent RNA analysis such as quantification, normalization, and visualization.

5.2.3 Features:

Reads count

bw file ->count file

Normalization

count file ->RPM files
count file ->RPKM files

5.2.4 Example

nasap feature_assign --output_root ./tmp --forward_bw ./tmp/bw/forward.bw --reverse_bw ./tmp/bw/reverse.bw --gtf /home/meta_data/annotation/Homo_sapiens.GRCh38.93.gtf

Parameters

parameter	description
--forward_bw (Required)	bowtie2 index file.
--reverse_bw (Required)	Sample FASTQ(gz) file.
--gtf (Required)	gtf file.
--output_root (Optional)	Output root directory.

--gtf:
Download specie gtf annotation file from ensembl database. Please note that the downloaded file should be unpacked first.

5.2.5 Results

Count files

File	Directory
protein coding count	csv/protein_coding_baseCount.csv
protein coding gb count	csv/protein_coding_gb_count.csv
protein coding pp count	csv/protein_coding_pp_count.csv
lincRNA count	csv/lincRNA_baseCount.csv
lincRNA gene body count	csv/lincRNA_gb_count.csv
lincRNA proximal promoter count	csv/lincRNA_pp_count.csv

Normalization files

File	Directory
protein coding rpkm	csv/protein_coding_rpkm.csv
lincRNA rpkm	csv/lincRNA_rpkm.csv
protein coding rpm	csv/protein_coding_rpm.csv
lincRNA rpm	csv/lincRNA_rpm.csv

Expressed genes pie plot

The plot shows statistical results for lincRNA and protein-coding RNA expressed genes.

Expressed RNA distribution plot

The relative abundance of different RNA molecules is evaluated in the plot.

Gene expression on different chromosomes

The distribution of expressed genes across chromosomes can be visualized using a chromosomal distribution plot.