3.9 Alignment
of Sequence Data
to Reference Genome
3.9.1 Tools: Bowtie2
1. Run Bowtie2 to map reads to a reference genome. Bowtie2 can
be accessed on Galaxy Main under Genomics Analysis !
Mapping ! Bowtie2 (see Note 30).
2. Select the .FASTQ file that you would like to map.
3. Select whether the data are single-end or paired-end.
4. Given the option “Will you select a reference genome from
your history or use a built-in index?” select “Use a genome
from the history and build index” (see Note 31).
The Bowtie2 tool will take .FASTQ reads and a reference
genome (indexed) as inputs and will output a SAM/BAM file for
each set of reads. This is an alignment file that can be viewed using
the Integrated Genomics Viewer [25] or additional tools within
and outside of Galaxy. However, in order to achieve the ultimate
goal of determining differential gene expression, there is a need to
quantify, normalize, and compare the data.
3.10 Reconstruction
of the Transcriptome
3.10.1 Tools:
htseq-count, featureCounts
The output from Bowtie2 will be utilized to determine the location
of the sequenced reads. Building upon this, it will be necessary to
bin these reads by gene and count the number of reads associated
with each gene using a tool such as featureCounts [26] or htseqcount [27], which are both available on the Galaxy platform. These
tools will take the SAM/BAM output of Bowtie2 as an input, as
well as the .GTF or .GFF genome annotation file that was mentioned above. It is critical that the annotation file is formatted
appropriately and that the annotations match what is in the
.FASTA file. To view the .GFF/.GTF file once it is uploaded,
click the eye icon. The ‘Feature’ and ‘Group’ columns are of
particular importance, as we will need to designate which feature
and which identifier for the tool to use when assigning mapped
reads to transcripts to determine a count. For this example, we will
use featureCounts and assume the use of .GTF and .FASTA files
that were downloaded from NCBI’s Genbank “Assembly”
database.
1. Access the featureCounts software on Galaxy Main under
Genomics Analysis ! RNA-seq ! featureCounts.
2. Select the outputs from the Bowtie2 mapping (Subheading
3.9) for the alignment file(s).
3. Specify strand information: Unstranded.
4. For the Gene annotation file, select the M. tuberculosis .GTF
file uploaded from Genbank from “in your history.”
5. Under Advanced options select the eye to show additional
options.
(a) GFF feature type filter should be “gene,”
(b) GFF gene identifier should be “gene_id,” and.
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Shuyi Ma et al.
of Sequence Data
to Reference Genome
3.9.1 Tools: Bowtie2
1. Run Bowtie2 to map reads to a reference genome. Bowtie2 can
be accessed on Galaxy Main under Genomics Analysis !
Mapping ! Bowtie2 (see Note 30).
2. Select the .FASTQ file that you would like to map.
3. Select whether the data are single-end or paired-end.
4. Given the option “Will you select a reference genome from
your history or use a built-in index?” select “Use a genome
from the history and build index” (see Note 31).
The Bowtie2 tool will take .FASTQ reads and a reference
genome (indexed) as inputs and will output a SAM/BAM file for
each set of reads. This is an alignment file that can be viewed using
the Integrated Genomics Viewer [25] or additional tools within
and outside of Galaxy. However, in order to achieve the ultimate
goal of determining differential gene expression, there is a need to
quantify, normalize, and compare the data.
3.10 Reconstruction
of the Transcriptome
3.10.1 Tools:
htseq-count, featureCounts
The output from Bowtie2 will be utilized to determine the location
of the sequenced reads. Building upon this, it will be necessary to
bin these reads by gene and count the number of reads associated
with each gene using a tool such as featureCounts [26] or htseqcount [27], which are both available on the Galaxy platform. These
tools will take the SAM/BAM output of Bowtie2 as an input, as
well as the .GTF or .GFF genome annotation file that was mentioned above. It is critical that the annotation file is formatted
appropriately and that the annotations match what is in the
.FASTA file. To view the .GFF/.GTF file once it is uploaded,
click the eye icon. The ‘Feature’ and ‘Group’ columns are of
particular importance, as we will need to designate which feature
and which identifier for the tool to use when assigning mapped
reads to transcripts to determine a count. For this example, we will
use featureCounts and assume the use of .GTF and .FASTA files
that were downloaded from NCBI’s Genbank “Assembly”
database.
1. Access the featureCounts software on Galaxy Main under
Genomics Analysis ! RNA-seq ! featureCounts.
2. Select the outputs from the Bowtie2 mapping (Subheading
3.9) for the alignment file(s).
3. Specify strand information: Unstranded.
4. For the Gene annotation file, select the M. tuberculosis .GTF
file uploaded from Genbank from “in your history.”
5. Under Advanced options select the eye to show additional
options.
(a) GFF feature type filter should be “gene,”
(b) GFF gene identifier should be “gene_id,” and.
506
Shuyi Ma et al.