© 2022 Janghyun Choi
Alignment with Reference Genome using bowtie2
Bowtie2 that is an ultrafast and memory-efficient tool coded in python can be aligned sequencing reads to long reference genomes. Notably, Bowtie is suitable for ChIP-seq or ATAC-seq, but not RNA-seq. If you have to analyze transcriptome, go to the section on RNA-seq using HISAT2 tool. Bowtie can run on any computer installed on Linux or macOS and works in python version > 2.6. This protocol was created based on Bowtie2 version 2.5.1 running on a system equipped with an Intel 10th generation i9-10910 processor and 48GB of memory. The test environment includes Python version 3.8.5, SciPy version 1.6.2, NumPy version 1.20.1, and pySam version 0.16.0.1 under macOS 12.4 environment. This course takes very long time and spends a lot of RAM and CPU resource.
Installation bowtie2
To install bowtie2 using homebrew, use the following command:
$ brew install bowtie2
Establish Genome Builder
bowtie2-build builds a bowtie index from a set of DNA sequence. This builder consists of a set of 6 files with suffixes .1.bt2, .2.bt2, .3.bt2, .4.bt2, .rev.1.bt2, and .rev.2.bt2. These files together constitute the index for the alignment reads to that reference. You can download from the index section of official website all the files for a given assembly as a single zip file, or as 6 seperate bt2 files. There are two types of genome builder method; manual and automatic.
- Automatic method: uncompress builder files and uses ‘bowtie2-build’ or ‘make_XX_.sh’ syntax. For more information, refer to Bowtie2’s manual.
- Manual method: uncompress builder files and moves them to the desired path.
the build step is a prerequisite for the main sequence. Unlike “HISAT2”, this tool provides index files for most species from the index zone of official site.
Running bowtie2
Use the following command to perform mapping to the genome with bowtie2:
# Usage for single-end
$ bowtie2 -x <GenomeBuilder> -U <reads.fq> -S <output.sam> --<mode> --<preset> -p <int>
# Usage for pair-end
$ bowtie2 -x <GenomeBuilder> -1 <forward_reads.fq> -2 <reverse_reads.fq> \
-S <output.sam> --<mode> --<preset> -p <int>
In these commands,
| Parameter | Description |
|---|---|
-x <GenomeBuilder> | Specifies the reference genome as the builder format. |
-U <reads.fq> (only SE mode) | Specifies input read sequence. Possible file types is fastq formats (fastq or fq). |
-1 and -2 (only PE mode) | Specifies forward input read and reverse input read, respectively. |
-S <output.sam> | Specifies output file. |
--<mode>> option | Bowtie2 provides two algorithms for maaping reads to the genome. The end-to-end mode (--end-to-end, default) aligns the entire length of reads to the reference genome, utilizing all available information. In contrast, the local mode (--local) flexibly aligns only the best-matching segments of reads to the reference, which is especially useful when the quality of reads is uneven. For more information, refer to Bowtie2’s manual. This manual provides the local algorithm. |
--<preset> option | Specifies preset options for the mapping algorithm. Possivle presers for the local mode are --very-fast-local, --fast-local, --sensitive-local (default) or --very-sensitive-local. For more information, refer to Bowtie2’s manual. |
-p <int> | number of processors to use in addition to main thread |
Example Code
Here is an example command to perform alignment with the mouse mm10 genome on trimmed fastq files:
$ bowtie2 -x /Users/jchoi/Desktop/mm10/mm10 \
-1 /Users/jchoi/Desktop/Trim/Trim_pair_NFIB_1.fq.gz \
-2 /Users/jchoi/Desktop/Trim/Trim_pair_NFIB_2.fq.gz \
-S /Users/jchoi/Desktop/NFIB.sam --local --very-fast-local -p 20
Output
When bowtie2 finishes running, it prints messages summarizing what happened.
23156585 reads; of these:
23156585 (100.00%) were paired; of these:
2044536 (8.83%) aligned concordantly 0 times
11769262 (50.82%) aligned concordantly exactly 1 time
9342787 (40.35%) aligned concordantly >1 times
----
2044536 pairs aligned concordantly 0 times; of these:
686243 (33.56%) aligned discordantly 1 time
----
1358293 pairs aligned 0 times concordantly or discordantly; of these:
2716586 mates make up the pairs; of these:
1327258 (48.86%) aligned 0 times
453087 (16.68%) aligned exactly 1 time
936241 (34.46%) aligned >1 times
97.13% overall alignment rate
- This message can also be outputted by
samtools.
The subsequent process utilizes the resulting ‘SAM’ file.