genomic alignment and finally a spliced alignment based on the available annotations
(Fig. 11.4a). The main steps using TopHat2 are summarized below:
Building Reference Index First, the reference genome of choice is indexed using
Bowtie2. The generated index file and genomic FASTA file are then used for the genomic
alignment of the reads.
Description of parameters:
-f
the path to genome Fasta file
-p
to launch a specified number of parallel threads
Read Alignment/Mapping Quality checked reads in FASTQ format or plain FASTA files
are accepted as input for TopHat2. TopHat2 is able to handle either single-end or pairedend reads and is able to integrate single-end reads into a paired-end alignment. Separate
commands are used for utilizing genome index and transcriptome index files. Note, for an
optimal pair-end alignment it is crucial to keep the same order of reads in the two files.
Availability of genomic annotations in the GTF/GFF file format could be utilized for initial
transcriptome alignment. Transcriptome alignment involves the creation of a transcriptome
index utilizing the genome index and the annotation information from GTF/GFF file.
The alignment using TopHat2 is performed as follows:
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