Bowtie

Bowtie (read mapper)

• Author: Dreycey Albin
• Date: 05/10/2019
• Updates: 05/12/2019 -- finished
• Mapping short sequences on to bigger sequences
• documentation (website): URL TO WEBSITE
• documentation (manual): http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml
• documentation (publication): BOWTIE 2

Installation

INSTRUCTIONS ON HOW TO INSTALL USING TERMINAL

• installation for MacOS

conda install bowtie2

OR

docker

OR

wget https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/bowtie2-2.3.5.1-macos-x86_64.zip/download;

• installation for Linux

conda install bowtie2;

OR

docker;

OR

wget https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/bowtie2-2.3.5.1-linux-x86_64.zip/download;

• installation from source

wget [bowtie2-2.3.5.1-source.zip](https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/bowtie2-2.3.5.1-source.zip/download "Click to download bowtie2-2.3.5.1-source.zip");

or try..

wget https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/bowtie2-2.3.5.1-sra-linux-x86_64.zip/download;

Basic commands

INPUT

• building the index

bowtie2-build [options]* <reference_in> <bt2_base>

• aligning the reads

bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i> | --sra-acc <acc> | b <bam>} -S [<sam>]

OUTPUT

PUT FILES HERE
GENERAL OUTPUT FROM PROGRAM (files and commands)

General notes:

• may do

  • end-to-end
  • local alignments

• Scoring techniques

  • End-to-end alignment score
  • Local alignment score

• There are thresholds for each scoring method

  • minimum score can be changed
    • --score-min flag

• mapping quality metric

  • can be thought of as uniqueness
  • Q = -10 log([p]), where p is probability that alignment does not correspond to reads true point of origin.

• concordance give an estimate on the paired end reads

  • must be within a certain distance from each other and expected orientation
  • This can be used to identify structural rearrangements

Example_1

Input files

• Build the index for the illumina example

bowtie2-build -f  -c GCF_000006765.1_ASM676v1_genomic.fna illumina_output

• Build the index

bowtie2-build -f  -c GCF_000005845.2_ASM584v2_genomic.fna output

output

output.1.bt2  
output.2.bt2  
output.3.bt2  
output.4.bt2  
output.rev.1.bt2  
output.rev.2.bt2

• Run Bowtie2

commands

• build the index

bowtie2-build -f GCF_000005845.2_ASM584v2_genomic.fna illumina_output_2

• next test out bowtie2

bowtie2 -p 70 -x illumina_output_2 -1 paired_dat1.fq -2 paired_dat2.fq -S albinout.sam

Output files

• index files

illumina_output_2.1.bt2  
illumina_output_2.3.bt2  
illumina_output_2.rev.1.bt2
illumina_output_2.2.bt2  
illumina_output_2.4.bt2  
illumina_output_2.rev.2.bt2

• Main output

albinout.sam

Example 2:

Input files

• obtain reference genome

wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/006/765/GCF_000006765.1_ASM676v1/GCF_000006765.1_ASM676v1_genomic.fna.gz;
gzip -d GCF_000006765.1_ASM676v1_genomic.fna.gz

• get illumina reads

wget http://resources.qiagenbioinformatics.com/testdata/paeruginosa-reads.zip;

Commands

• build index

bowtie2-build -f GCF_000006765.1_ASM676v1_genomic.fna illumina_output_test

• test out read mapping

bowtie2 -p 70 -x illumina_output_test -1 paeruginosa-reads/SRR396636.sra_1.fastq -2 paeruginosa-reads/SRR396636.sra_2.fastq -S illumina_test_out.sam

• convert sam to bam

./../samtools/samtools view -S -b illumina_test_out.sam > illumina.bam

• view the output

samtools view sample.bam | head

• sort the BAM file

./../samtools/samtools sort illumina.bam -o illumina.sorted.bam

*view the output

samtools view illumina.sorted.bam | head

Output files

• index files

\illumina_output_test.1.bt2  
illumina_output_test.3.bt2  
illumina_output_test.rev.1.bt2
illumina_output_test.2.bt2  
illumina_output_test.4.bt2  
illumina_output_test.rev.2.bt2

• main output

illumina.bam
illumina.sorted.bam

From bowtie website

Paired-end example

To align paired-end reads included with Bowtie 2, stay in the same directory and run:

$BT2_HOME/bowtie2 -x lambda_virus -1 $BT2_HOME/example/reads/reads_1.fq -2 $BT2_HOME/example/reads/reads_2.fq -S eg2.sam

This aligns a set of paired-end reads to the reference genome, with results written to the file eg2.sam.

Local alignment example

To use local alignment to align some longer reads included with Bowtie 2, stay in the same directory and run:

$BT2_HOME/bowtie2 --local -x lambda_virus -U $BT2_HOME/example/reads/longreads.fq -S eg3.sam

This aligns the long reads to the reference genome using local alignment, with results written to the file eg3.sam.

Paramaters

website : parameters for Bowtie

### Main arguments

-x <bt2-idx>
The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final  `.1.bt2`/  `.rev.1.bt2`  / etc.  `bowtie2`  looks for the specified index first in the current directory, then in the directory specified in the  `BOWTIE2_INDEXES`environment variable.

-1 <m1>
Comma-separated list of files containing mate 1s (filename usually includes  `_1`), e.g. `-1 flyA_1.fq,flyB_1.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in  `<m2>`. Reads may be a mix of different lengths. If  `-`  is specified,  `bowtie2`will read the mate 1s from the “standard in” or “stdin” filehandle.

-2 <m2>
Comma-separated list of files containing mate 2s (filename usually includes  `_2`), e.g. `-2 flyA_2.fq,flyB_2.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in  `<m1>`. Reads may be a mix of different lengths. If  `-`  is specified,  `bowtie2`will read the mate 2s from the “standard in” or “stdin” filehandle.

-U <r>
Comma-separated list of files containing unpaired reads to be aligned, e.g.  `lane1.fq,lane2.fq,lane3.fq,lane4.fq`. Reads may be a mix of different lengths. If  `-`  is specified,  `bowtie2`  gets the reads from the “standard in” or “stdin” filehandle.

--interleaved
Reads interleaved FASTQ files where the first two records (8 lines) represent a mate pair.

--sra-acc
Reads are SRA accessions. If the accession provided cannot be found in local storage it will be fetched from the NCBI database. If you find that SRA alignments are long running please rerun your command with the  [`-p`/`--threads`](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#bowtie2-options-p)  parameter set to desired number of threads.

NB: this option is only available if bowtie 2 is compiled with the necessary SRA libraries. See  [Obtaining Bowtie 2](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#obtaining-bowtie-2)  for details.

-b <bam>
Reads are unaligned BAM records sorted by read name. The  [`--align-paired-reads`](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#bowtie2-options-align-paired-reads)  and  [`--preserve-tags`](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#bowtie2-options-preserve-tags)  options affect the way Bowtie 2 processes records.

-S <sam>
File to write SAM alignments to. By default, alignments are written to the “standard out” or “stdout” filehandle (i.e. the console).