Example of creating a bowtie2-index based on E. coli reference genomes. cat ref_genomes/ecoli/*.fna > genomes.fna bowtie2-build genomes.fna ecoli # result: 6 .bt2 database files ls ecoli.1.bt2 ecoli.2.bt2 ecoli.3.bt2 ecoli.4.bt2 ecoli.rev.1.bt2 ecoli.rev.2.bt2 # use 'inspect' to check the content of your database bowtie2-inspect -n ecoli # define your BOWTIE2_INDEXES directory and move the database files into it export BOWTIE2_INDEXES=/path/to/my/bowtie2/databases/ cp *.bt2 $BOWTIE2_INDEXES # in bowtie2 mapping, database is defined by option -x bowtie2 -x ecoli ... |