Package 'MicroSEC' reference manual

Title:	Sequence Error Filter for Formalin-Fixed and Paraffin-Embedded Samples
Description:	Clinical sequencing of tumor is usually performed on formalin-fixed and paraffin-embedded samples and have many sequencing errors. We found that the majority of these errors are detected in chimeric read caused by single-strand DNA with micro-homology. Our filtering pipeline focuses on the uneven distribution of the artifacts in each read and removes such errors in formalin-fixed and paraffin-embedded samples without over-eliminating the true mutations detected in fresh frozen samples.
Authors:	Masachika Ikegami [aut, cre]
Maintainer:	Masachika Ikegami <[email protected]>
License:	MIT + file LICENSE
Version:	2.1.6
Built:	2024-11-26 05:59:16 UTC
Source:	https://github.com/mano-b/microsec

An example BAM file.

Description

A BAM file containing the information of eight mutations.

Usage

exampleBam
exampleBam

Format

A list with 8 factors, each contains 46527 variables:

rname: chromosome of the read
qname: read ID list
seq: sequence of the read, in DNAString
strand: strand of the read
cigar: CIGAR sequence of the read
qual: Phred quality of the read
pos: starting position of the read
isize: insert size of the read

...

An example mutation file.

Description

A dataset containing the information of eight mutations.

Usage

exampleMutation
exampleMutation

Format

A list with 8 factors, each contains 29 variables

Sample: sample name
Mut_type: mutation type
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
SimpleRepeat_TRF: mutation locating repeat sequence
Neighborhood_sequence: neighborhood sequence

...

Analyzing function.

Description

This function analyzes the filtering results.

Usage

fun_analysis(
  msec,
  mut_depth,
  short_homology_search_length,
  min_homology_search,
  threshold_p,
  threshold_hairpin_ratio,
  threshold_short_length,
  threshold_distant_homology,
  threshold_soft_clip_ratio,
  threshold_low_quality_rate,
  homopolymer_length
)
fun_analysis(
  msec,
  mut_depth,
  short_homology_search_length,
  min_homology_search,
  threshold_p,
  threshold_hairpin_ratio,
  threshold_short_length,
  threshold_distant_homology,
  threshold_soft_clip_ratio,
  threshold_low_quality_rate,
  homopolymer_length
)

Arguments

`msec`	Mutation filtering information.
`mut_depth`	Mutation coverage data.
`short_homology_search_length`	Small sequence for homology search.
`min_homology_search`	The sequence length for homology search.
`threshold_p`	The largest p value of significant errors.
`threshold_hairpin_ratio`	The smallest hairpin read ratio.
`threshold_short_length`	Reads shorter than that are analyzed.
`threshold_distant_homology`	The smallest rate of reads from other regions.
`threshold_soft_clip_ratio`	The rate of soft-clipped reads.
`threshold_low_quality_rate`	The smallest rate of low quality bases.
`homopolymer_length`	The smallest length of homopolymers.

Value

msec

Examples

data(msec_summarized)
data(mut_depth_checked)
fun_analysis(msec = msec_summarized,
             mut_depth = mut_depth_checked,
             short_homology_search_length = 4,
             min_homology_search = 40,
             threshold_p = 10 ^ (-6),
             threshold_hairpin_ratio = 0.50,
             threshold_short_length = 0.75,
             threshold_distant_homology = 0.15,
             threshold_soft_clip_ratio = 0.50,
             threshold_low_quality_rate = 0.1,
             homopolymer_length = 15
)
data(msec_summarized)
data(mut_depth_checked)
fun_analysis(msec = msec_summarized,
             mut_depth = mut_depth_checked,
             short_homology_search_length = 4,
             min_homology_search = 40,
             threshold_p = 10 ^ (-6),
             threshold_hairpin_ratio = 0.50,
             threshold_short_length = 0.75,
             threshold_distant_homology = 0.15,
             threshold_soft_clip_ratio = 0.50,
             threshold_low_quality_rate = 0.1,
             homopolymer_length = 15
)

Hairpin-structure sequence check function

Description

This function attempts to find hairpin structure sequences.

Usage

fun_hairpin_check(hairpin_seq_tmp, ref_seq, hairpin_length, hair)
fun_hairpin_check(hairpin_seq_tmp, ref_seq, hairpin_length, hair)

Arguments

`hairpin_seq_tmp`	The sequence to be checked.
`ref_seq`	Reference sequence around the mutation.
`hairpin_length`	The temporal length of hairpin sequences.
`hair`	The length of sequences to be checked.

Value

list(hairpin_length, whether hairpin sequences exist or not)

Homology check function.

Description

This function attempts to search the homologous regions.

Usage

fun_homology(
  msec,
  df_distant,
  min_homology_search,
  ref_genome,
  chr_no,
  progress_bar
)
fun_homology(
  msec,
  df_distant,
  min_homology_search,
  ref_genome,
  chr_no,
  progress_bar
)

Arguments

`msec`	Mutation filtering information.
`df_distant`	Sequences to be checked.
`min_homology_search`	Minimum length to define "homologous".
`ref_genome`	Reference genome for the data.
`chr_no`	Reference genome chromosome number (human=24, mouse=22).
`progress_bar`	"Y": You can see the progress visually.

Value

msec

Examples

## Not run: 
data(msec_read_checked)
data(homology_searched)
fun_homology(msec = msec_read_checked,
   df_distant = homology_searched,
   min_homology_search = 40,
   ref_genome = BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
   chr_no = 24,
   progress_bar = "N"
)

## End(Not run)
## Not run: 
data(msec_read_checked)
data(homology_searched)
fun_homology(msec = msec_read_checked,
   df_distant = homology_searched,
   min_homology_search = 40,
   ref_genome = BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
   chr_no = 24,
   progress_bar = "N"
)

## End(Not run)

BAM file loader

Description

This function attempts to load the BAM file.

Usage

fun_load_bam(bam_file)
fun_load_bam(bam_file)

Arguments

bam_file

Path of the BAM file.

Value

df_bam

Examples

fun_load_bam(
  system.file("extdata", "sample.bam", package = "MicroSEC")
)
fun_load_bam(
  system.file("extdata", "sample.bam", package = "MicroSEC")
)

Chromosome number loading function.

Description

This function attempts to load the chromosome number.

Usage

fun_load_chr_no(organism)
fun_load_chr_no(organism)

Arguments

organism

Human or Mouse genome.

Value

chr_no

Examples

fun_load_chr_no("Human")
fun_load_chr_no("Human")

Genome loading function.

Description

This function attempts to load the appropriate genome.

Usage

fun_load_genome(organism)
fun_load_genome(organism)

Arguments

organism

Human or Mouse genome.

Value

ref_genome

Examples

fun_load_genome("Human")
fun_load_genome("Human")

Mutation data file loader

Description

This function attempts to load the mutation information file.

Usage

fun_load_mutation(
  mutation_file,
  sample_name,
  ref_genome,
  chr_no,
  simple_repeat_list = ""
)
fun_load_mutation(
  mutation_file,
  sample_name,
  ref_genome,
  chr_no,
  simple_repeat_list = ""
)

Arguments

`mutation_file`	Path of the mutation information file.
`sample_name`	Sample name.
`ref_genome`	Reference genome for the data.
`chr_no`	Reference genome chromosome number (human=24, mouse=22).
`simple_repeat_list`	Optional, set simple repeat bed file path.

Value

df_mutation

Examples

fun_load_mutation(
  system.file("extdata", "mutation_list.tsv", package = "MicroSEC"),
  "sample",
  BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
  24
)
fun_load_mutation(
  system.file("extdata", "mutation_list.tsv", package = "MicroSEC"),
  "sample",
  BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
  24
)

Read check function.

Description

This function attempts to check the mutation profile in each read.

Usage

fun_read_check(
  df_mutation,
  df_bam,
  ref_genome,
  sample_name,
  read_length,
  adapter_1,
  adapter_2,
  short_homology_search_length,
  min_homology_search,
  progress_bar
)
fun_read_check(
  df_mutation,
  df_bam,
  ref_genome,
  sample_name,
  read_length,
  adapter_1,
  adapter_2,
  short_homology_search_length,
  min_homology_search,
  progress_bar
)

Arguments

`df_mutation`	Mutation information.
`df_bam`	Data from the BAM file.
`ref_genome`	Reference genome for the data.
`sample_name`	Sample name (character)
`read_length`	The read length in the sequence.
`adapter_1`	The Read 1 adapter sequence of the library.
`adapter_2`	The Read 2 adapter sequence of the library.
`short_homology_search_length`	Small sequence for homology search.
`min_homology_search`	Minimum length to define "homologous".
`progress_bar`	"Y": You can see the progress visually.

Value

list(msec, homology_search, mut_depth)

Examples

## Not run: 
data(exampleMutation)
data(exampleBam)
fun_read_check(df_mutation = exampleMutation,
df_bam = exampleBam,
ref_genome = BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
sample_name = "sample",
read_length = 150,
adapter_1 = "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA",
adapter_2 = "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT",
short_homology_search_length = 4,
min_homology_search = 40,
progress_bar = "N"
)

## End(Not run)
## Not run: 
data(exampleMutation)
data(exampleBam)
fun_read_check(df_mutation = exampleMutation,
df_bam = exampleBam,
ref_genome = BSgenome.Hsapiens.UCSC.hg38::BSgenome.Hsapiens.UCSC.hg38,
sample_name = "sample",
read_length = 150,
adapter_1 = "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA",
adapter_2 = "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT",
short_homology_search_length = 4,
min_homology_search = 40,
progress_bar = "N"
)

## End(Not run)

Repeat check function.

Description

This function attempts to check the repetitive sequence around the mutation.

Usage

fun_repeat_check(rep_a, rep_b, ref_seq, ref_width, del)
fun_repeat_check(rep_a, rep_b, ref_seq, ref_width, del)

Arguments

`rep_a`	The shorter sequence of Ref and Alt.
`rep_b`	The longer sequence of Ref and Alt.
`ref_seq`	Reference sequence around the mutation.
`ref_width`	Search length for ref_seq.
`del`	Insertion: 0, Deletion: 1

Value

list(pre_rep_status, post_rep_status, pre_rep_short, post_rep_short, homopolymer_status)

Save function.

Description

This function attempts to save the filtering results.

Usage

fun_save(msec, output)
fun_save(msec, output)

Arguments

`msec`	Mutation filtering information.
`output`	output file name (full path).

Examples

## Not run: 
data(msec_analyzed)
fun_save(msec = msec_analyzed,
         output = "./MicroSEC_test.tsv.gz"
)

## End(Not run)
## Not run: 
data(msec_analyzed)
fun_save(msec = msec_analyzed,
         output = "./MicroSEC_test.tsv.gz"
)

## End(Not run)

Mutated position search function.

Description

This function attempts to find the mutated bases in each read.

Usage

fun_setting(pre, post, neighbor_seq, neighbor_length, alt_length)
fun_setting(pre, post, neighbor_seq, neighbor_length, alt_length)

Arguments

`pre`	The 5' side bases of the sequence for searching.
`post`	The 3' side bases of the sequence for searching.
`neighbor_seq`	Short reference sequence around the mutation.
`neighbor_length`	The length from the mutation to the ends of the short reference sequence.
`alt_length`	The length of altered bases.

Value

list(pre_search_length, post_search_length, peri_seq_1, peri_seq_2)

Summarizing function.

Description

This function summarizes the filtering results.

Usage

fun_summary(msec)
fun_summary(msec)

Arguments

msec

Mutation filtering information.

Value

msec

Examples

data(msec_homology_searched)
fun_summary(msec_homology_searched)
data(msec_homology_searched)
fun_summary(msec_homology_searched)

Divide function without 0/0 errors

Description

This function attempts to divide without 0/0 errors.

Usage

fun_zero(a, b)
fun_zero(a, b)

Arguments

a, b

Integers

Value

a divided by b

An example sequence information file.

Description

A dataset containing the information of reads for homology search.

Usage

homology_searched
homology_searched

Format

A list with 7 factors, each contains 1508 variables:

sample_name: sample name
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
Direction: 5' (pre) or 3' (post) sequence of the mutated base
Seq: sequence for homology search

...

An example mutation file.

Description

A dataset containing the information of eight mutations processed by the fun_homology function.

Usage

msec_analyzed
msec_analyzed

Format

A list with 37 factors, each contains 29 variables

Sample: sample name
Mut_type: mutation type
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
SimpleRepeat_TRF: mutation locating repeat sequence
Neighborhood_sequence: neighborhood sequence
read_length: read length
total_read: number of mutation supporting reads
soft_clipped_read: number of soft-clipped reads
flag_hairpin: number of reads produced by hairpin structure
pre_support_length: maximum 5'-supporting length
post_support_length: maximum 3'-supporting length
short_support_length: minimum supporting length
pre_farthest: 5'-farthest supported base from the mutated base
post_farthest: 3'-farthest supported base from the mutated base
low_quality_base_rate_under_q18: low quality base rate
low_quality_pre: low quality base rate of 5'- side
low_quality_post: low quality base rate of 3'- side
distant_homology_rate: rate of reads derived from homologous regions
soft_clipped_rate: rate of soft clipped reads
prob_filter_1: possibility of short-supporting length
prob_filter_3_pre: possibility of 5'-supporting length
prob_filter_3_post: possibility of 3'-supporting length
filter_1_mutation_intra_hairpin_loop: filter 1
filter_2_hairpin_structure: filter 2
filter_3_microhomology_induced_mutation: filter 3
filter_4_highly_homologous_region: filter 4
filter_5_soft_clipping: filter 5
filter_6_simple_repeat: filter 6
filter_7_mutation_at_homopolymer: filter 7
filter_8_low_quality: filter 8
msec_filter_123: any of filter 1-3
msec_filter_1234: any of filter 1-4
msec_filter_all: any of filter 1-8
comment: comment

...

An example mutation file.

Description

A dataset containing the information of eight mutations processed by the fun_homology function.

Usage

msec_homology_searched
msec_homology_searched

Format

A list with 34 factors, each contains 29 variables

Sample: sample name
Mut_type: mutation type
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
SimpleRepeat_TRF: mutation locating repeat sequence
Neighborhood_sequence: neighborhood sequence
read_length: read length
mut_type: mutation type
alt_length: length of the mutated bases
total_read: number of mutation supporting reads
soft_clipped_read: number of soft-clipped reads
flag_hairpin: number of reads produced by hairpin structure
hairpin_length: maximum length of palindromic sequences
pre_support_length: maximum 5'-supporting length
post_support_length: maximum 3'-supporting length
short_support_length: minimum supporting length
pre_minimum_length: minimum 5'-supporting length
post_minimum_length: minimum 3'-supporting length
pre_farthest: 5'-farthest supported base from the mutated base
post_farthest: 3'-farthest supported base from the mutated base
low_quality_base_rate_under_q18: low quality base rate
low_quality_pre: low quality base rate of 5'- side
low_quality_post: low quality base rate of 3'- side
pre_rep_status: 5'-repeat sequence length
post_rep_status: 3'-repeat sequence length
homopolymer_status: homopolymer sequence length
indel_status: whether the mutation is indel or not
indel_length: length of indel mutation
distant_homology: number of reads derived from homologous regions
penalty_pre: 5'-penalty score by the mapper
penalty_post: 3'-penalty score by the mapper
caution: comment

...

An example mutation file.

Description

A dataset containing the information of eight mutations processed by the fun_read_check function.

Usage

msec_read_checked
msec_read_checked

Format

A list with 34 factors, each contains 46527 variables

Sample: sample name
Mut_type: mutation type
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
SimpleRepeat_TRF: mutation locating repeat sequence
Neighborhood_sequence: neighborhood sequence
read_length: read length
mut_type: mutation type
alt_length: length of the mutated bases
total_read: number of mutation supporting reads
soft_clipped_read: number of soft-clipped reads
flag_hairpin: number of reads produced by hairpin structure
hairpin_length: maximum length of palindromic sequences
pre_support_length: maximum 5'-supporting length
post_support_length: maximum 3'-supporting length
short_support_length: minimum supporting length
pre_minimum_length: minimum 5'-supporting length
post_minimum_length: minimum 3'-supporting length
pre_minimum_length: minimum 5'-supporting length
low_quality_base_rate_under_q18: low quality base rate
low_quality_pre: low quality base rate of 5'- side
low_quality_post: low quality base rate of 3'- side
pre_farthest: 5'-farthest supported base from the mutated base
post_farthest: 3'-farthest supported base from the mutated base
post_rep_status: 3'-repeat sequence length
homopolymer_status: homopolymer sequence length
indel_status: whether the mutation is indel or not
indel_length: length of indel mutation
distant_homology: number of reads derived from homologous regions
penalty_pre: 5'-penalty score by the mapper
penalty_post: 3'-penalty score by the mapper
caution: comment

...

An example mutation file.

Description

A dataset containing the information of eight mutations processed by the fun_homology function.

Usage

msec_summarized
msec_summarized

Format

A list with 52 factors, each contains 29 variables

Sample: sample name
Mut_type: mutation type
Chr: altered chromosome
Pos: altered position
Ref: reference base
Alt: altered base
SimpleRepeat_TRF: mutation locating repeat sequence
Neighborhood_sequence: neighborhood sequence
read_length: read length
mut_type: mutation type
alt_length: length of the mutated bases
total_read: number of mutation supporting reads
soft_clipped_read: number of soft-clipped reads
flag_hairpin: number of reads produced by hairpin structure
hairpin_length: maximum length of palindromic sequences
pre_support_length: maximum 5'-supporting length
post_support_length: maximum 3'-supporting length
short_support_length: minimum supporting length
pre_minimum_length: minimum 5'-supporting length
post_minimum_length: minimum 3'-supporting length
pre_farthest: 5'-farthest supported base from the mutated base
post_farthest: 3'-farthest supported base from the mutated base
low_quality_base_rate_under_q18: low quality base rate
low_quality_pre: low quality base rate of 5'- side
low_quality_post: low quality base rate of 3'- side
pre_rep_status: 5'-repeat sequence length
post_rep_status: 3'-repeat sequence length
homopolymer_status: homopolymer sequence length
indel_status: whether the mutation is indel or not
indel_length: length of indel mutation
distant_homology: number of reads derived from homologous regions
penalty_pre: 5'-penalty score by the mapper
penalty_post: 3'-penalty score by the mapper
caution: comment
distant_homology_rate: rate of reads derived from homologous regions
pre_minimum_length_adj: adjusted pre_minimum_length
post_minimum_length_adj: adjusted pre_minimum_length
pre_support_length_adj: adjusted pre_minimum_length
post_support_length_adj: adjusted pre_minimum_length
shortest_support_length_adj: the shortest short_support_length
minimum_length_1: theoretically minimum 5'-supporting length
minimum_length_2: theoretically minimum 3'-supporting length
minimum_length: theoretically minimum supporting length
short_support_length_adj: adjusted short_support_length
altered_length: substituted/inserted length
half_length: half of the read length
short_support_length_total: range of short_support_length
pre_support_length_total: range of pre_support_length
post_support_length_total: range of post_support_length
half_length_total: range of possible short_support_length
total_length_total: range of possible supporting length
soft_clipped_rate: rate of soft clipped reads

...

An example sequence information file.

Description

A dataset containing the information of reads for homology search.

Usage

mut_depth_checked
mut_depth_checked

Format

Three lists with 201 factors, each contains 29 variables:

Package 'MicroSEC'

Help Index

An example BAM file.

Description

Usage

Format

An example mutation file.

Description

Usage

Format

Analyzing function.

Description

Usage

Arguments

Value

Examples

Hairpin-structure sequence check function

Description

Usage

Arguments

Value

Homology check function.

Description

Usage

Arguments

Value

Examples

BAM file loader

Description

Usage

Arguments

Value

Examples

Chromosome number loading function.

Description

Usage

Arguments

Value

Examples

Genome loading function.

Description

Usage

Arguments

Value

Examples

Mutation data file loader

Description

Usage

Arguments

Value

Examples

Read check function.

Description

Usage

Arguments

Value

Examples

Repeat check function.

Description

Usage

Arguments

Value

Save function.

Description

Usage

Arguments

Examples

Mutated position search function.

Description

Usage

Arguments

Value

Summarizing function.

Description

Usage

Arguments

Value

Examples

Divide function without 0/0 errors

Description