Skip to content

Index Generation

Following bisulfite conversion, the sense (Watson) and anti-sense (Crick) reference strands are no longer complementary. Correctly aligning bisulfite converted reads requires the construction of an alignment index containing a bisulfite converted sequence for each reference strand.

BSB support generation of 3 types of alignment indices.

  1. Whole genome bisulfite sequencing indices
    • Index is generated for complete Watson and Crick strands
  2. Masked alignment indices
    • Sequence outside the reference region is masked in both the Watson and Crick strands before index generation
  3. Reduced representation bisulfite sequencing (RRBS) indices
    • The reference sequence is In silico restriction enzyme digested to give a list of plausible target regions

BSB Index Commands

bsbolt Index -G {fasta reference} -DB {database output}

-h, --help     show this help message and exit

Index Options:
  -G File      path to reference genome fasta file, fasta file should contain all contigs
  -DB File     path to index directory, alignment index generated inside existing directory
               or new directory made if directory doesn't exist
  -B Int       block size for bwtsw algorithm,
               increasing block will speed up indexing and increase memory consumption [10000000]
  -MR File     path to bed file of mappable regions.
               Index will be built using using masked contig sequence [null]
  -IA          ignore alt contigs when constructing alignment index
  -rrbs        generate a Reduced Representative Bisulfite Sequencing (RRBS) index

  -rrbs-cut-format Str    Cut format to use for generation of RRBS database, [C-CGG] MSPI,
                          input multiple enzymes as a comma separate string, C-CGG,C-CGG,...
  -rrbs-lower Int      lower bound fragment size to consider RRBS index generation [40]
  -rrbs-upper Int      upper bound fragment size to consider RRBS index generation [500]

WGBS Index Generation Example

python3 -m bsbolt Index -G ~/tests/TestData/BSB_test.fa -DB ~/tests/TestData/BSB_Test_DB

Masked Alignment Index Generation Example

python3 -m bsbolt Index -G ~/tests/TestData/BSB_test.fa -DB ~/tests/TestData/BSB_Test_DB -MR /tests/TestData/test_wgbs_madking.bed

RRBS Index Generation Example

# RRBS Index, MSPI Cut Format, 40bp Lower Fragment Bound, and 400bp Upper Fragment Bound
python3 -m bsbolt Index -G ~/tests/TestData/BSB_test.fa -DB ~/tests/TestData/BSB_Test_DB -rrbs -rrbs-cut-format C-CGG -rrbs-lower 40 -rrbs-upper 400