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use super::bamlift::*;
use super::*;
use bio::alphabets::dna::revcomp;
use colored::Colorize;
use lazy_static::lazy_static;
use rayon::{current_num_threads, prelude::*};
use regex::Regex;
use rust_htslib::{
bam, bam::ext::BamRecordExtensions, bam::record::Aux, bam::HeaderView, bam::Read,
};
use std::convert::TryFrom;
use std::fmt::Write;
use std::time::Instant;
pub struct BaseMod {
pub modified_base: u8,
pub strand: char,
pub modification_type: char,
modified_bases: Vec<i64>,
modified_bases_forward: Vec<i64>,
modified_probabilities: Vec<u8>,
reference_positions: Vec<i64>,
}
impl BaseMod {
pub fn new(
record: &bam::Record,
modified_base: u8,
strand: char,
modification_type: char,
modified_bases_forward: Vec<i64>,
modified_probabilities: Vec<u8>,
) -> Self {
let modified_bases = positions_on_complimented_sequence(record, &modified_bases_forward);
let reference_positions = get_exact_reference_positions(record, &modified_bases);
Self {
modified_base,
strand,
modification_type,
modified_bases,
modified_bases_forward,
modified_probabilities,
reference_positions,
}
}
pub fn get_reference_positions(&self) -> Vec<i64> {
self.reference_positions.clone()
}
pub fn get_modified_bases(&self) -> Vec<i64> {
self.modified_bases.clone()
}
pub fn get_modified_bases_forward(&self) -> Vec<i64> {
self.modified_bases_forward.clone()
}
pub fn get_modified_probabilities(&self) -> Vec<u8> {
if self.strand == '-' {
self.modified_probabilities
.clone()
.into_iter()
.rev()
.collect()
} else {
self.modified_probabilities.clone()
}
}
pub fn is_m6a(&self) -> bool {
self.modification_type == 'a'
}
pub fn is_cpg(&self) -> bool {
self.modification_type == 'm'
}
}
pub struct BaseMods {
pub base_mods: Vec<BaseMod>,
}
impl BaseMods {
pub fn new(record: &bam::Record, min_ml_score: u8) -> BaseMods {
lazy_static! {
static ref MM_RE: Regex =
Regex::new(r"((([ACGTUN])([-+])([a-z]+|[0-9]+))[.?]?((,[0-9]+)*;)*)").unwrap();
}
let mut rtn = vec![];
let ml_tag = get_u8_tag(record, b"ML");
let mut num_mods_seen = 0;
if let Ok(Aux::String(mm_text)) = record.aux(b"MM") {
for cap in MM_RE.captures_iter(mm_text) {
let mod_base = cap.get(3).map(|m| m.as_str().as_bytes()[0]).unwrap();
let mod_strand = cap.get(4).map_or("", |m| m.as_str());
let modification_type = cap.get(5).map_or("", |m| m.as_str());
let mod_dists_str = cap.get(6).map_or("", |m| m.as_str());
let mod_dists: Vec<i64> = mod_dists_str
.trim_end_matches(';')
.split(',')
.map(|s| s.trim())
.filter(|s| !s.is_empty())
.map(|s| s.parse().unwrap())
.collect();
let forward_bases = if record.is_reverse() {
revcomp(record.seq().as_bytes())
} else {
record.seq().as_bytes()
};
let mut cur_mod_idx = 0;
let mut cur_seq_idx = 0;
let mut dist_from_last_mod_base = 0;
let mut modified_positions: Vec<i64> = vec![0; mod_dists.len()];
while cur_seq_idx < forward_bases.len() && cur_mod_idx < mod_dists.len() {
let cur_base = forward_bases[cur_seq_idx];
if cur_base == mod_base && dist_from_last_mod_base == mod_dists[cur_mod_idx] {
modified_positions[cur_mod_idx] = i64::try_from(cur_seq_idx).unwrap();
dist_from_last_mod_base = 0;
cur_mod_idx += 1;
} else if cur_base == mod_base {
dist_from_last_mod_base += 1
}
cur_seq_idx += 1;
}
assert_eq!(cur_mod_idx, mod_dists.len());
let num_mods_cur_end = num_mods_seen + modified_positions.len();
let modified_probabilities = if num_mods_cur_end > ml_tag.len() {
let needed_num_of_zeros = num_mods_cur_end - ml_tag.len();
let mut to_add = vec![0; needed_num_of_zeros];
let mut has = ml_tag[num_mods_seen..ml_tag.len()].to_vec();
has.append(&mut to_add);
log::warn!(
"ML tag is too short for the number of modifications found in the MM tag. Assuming an ML value of 0 after the first {num_mods_cur_end} modifications."
);
has
} else {
ml_tag[num_mods_seen..num_mods_cur_end].to_vec()
};
num_mods_seen = num_mods_cur_end;
assert_eq!(modified_positions.len(), modified_probabilities.len());
let (modified_probabilities, modified_positions): (Vec<u8>, Vec<i64>) =
modified_probabilities
.iter()
.zip(modified_positions.iter())
.filter(|(&ml, &_mm)| ml >= min_ml_score)
.unzip();
let mods = BaseMod::new(
record,
mod_base,
mod_strand.chars().next().unwrap(),
modification_type.chars().next().unwrap(),
modified_positions,
modified_probabilities,
);
rtn.push(mods);
}
} else {
log::debug!("No MM tag found");
}
if ml_tag.len() > num_mods_seen {
log::warn!("ML tag has more entries than # of modifications in the MM tag.");
}
BaseMods { base_mods: rtn }
}
pub fn m6a_positions(&self, reference: bool) -> Vec<i64> {
let m6a: Vec<&BaseMod> = self.base_mods.iter().filter(|x| x.is_m6a()).collect();
if m6a.is_empty() {
return vec![];
}
if m6a.len() == 1 {
if reference {
return m6a[0].get_reference_positions();
} else {
return m6a[0].get_modified_bases();
}
}
if reference {
merge_two_lists(
&m6a[0].get_reference_positions(),
&m6a[1].get_reference_positions(),
)
} else {
merge_two_lists(&m6a[0].get_modified_bases(), &m6a[1].get_modified_bases())
}
}
pub fn m6a(&self, reference: bool) -> (Vec<i64>, Vec<u8>) {
let m6a: Vec<&BaseMod> = self.base_mods.iter().filter(|x| x.is_m6a()).collect();
if m6a.is_empty() {
return (vec![], vec![]);
}
let m6a_l = if reference {
m6a[0].get_reference_positions()
} else {
m6a[0].get_modified_bases()
};
let m6a_l_q = m6a[0].get_modified_probabilities();
let m6a_r;
let m6a_r_q;
if m6a.len() == 1 {
m6a_r = vec![];
m6a_r_q = vec![];
} else {
if reference {
m6a_r = m6a[1].get_reference_positions();
} else {
m6a_r = m6a[1].get_modified_bases();
}
m6a_r_q = m6a[1].get_modified_probabilities();
}
unzip_to_vectors(merge_two_lists_with_qual(
&m6a_l, &m6a_l_q, &m6a_r, &m6a_r_q,
))
}
pub fn cpg_positions(&self, reference: bool) -> Vec<i64> {
let cpg: Vec<&BaseMod> = self.base_mods.iter().filter(|x| x.is_cpg()).collect();
if cpg.is_empty() {
return vec![];
}
if reference {
cpg[0].get_reference_positions()
} else {
cpg[0].get_modified_bases()
}
}
}
pub fn get_u32_tag(record: &bam::Record, tag: &[u8; 2]) -> Vec<i64> {
if let Ok(Aux::ArrayU32(array)) = record.aux(tag) {
let read_array = array.iter().map(|x| x as i64).collect::<Vec<_>>();
read_array
} else {
vec![]
}
}
pub fn get_u8_tag(record: &bam::Record, tag: &[u8; 2]) -> Vec<u8> {
if let Ok(Aux::ArrayU8(array)) = record.aux(tag) {
let read_array = array.iter().collect::<Vec<_>>();
read_array
} else {
vec![]
}
}
pub struct FiberseqData {
pub record: bam::Record,
pub nuc: Vec<(i64, i64)>,
pub msp: Vec<(i64, i64)>,
pub ref_nuc: Vec<(i64, i64)>,
pub ref_msp: Vec<(i64, i64)>,
pub base_mods: BaseMods,
pub ec: f32,
}
impl FiberseqData {
pub fn new(record: &bam::Record, min_ml_score: u8) -> Self {
let mut nuc_starts = get_u32_tag(record, b"ns");
let mut msp_starts = get_u32_tag(record, b"as");
let mut nuc_length = get_u32_tag(record, b"nl");
let mut msp_length = get_u32_tag(record, b"al");
let mut nuc_ends = nuc_starts
.iter()
.zip(nuc_length.iter())
.map(|(&x, &y)| x + y)
.collect::<Vec<_>>();
let mut msp_ends = msp_starts
.iter()
.zip(msp_length.iter())
.map(|(&x, &y)| x + y)
.collect::<Vec<_>>();
if record.is_reverse() {
(nuc_ends, nuc_starts) = (
positions_on_complimented_sequence(record, &nuc_starts),
positions_on_complimented_sequence(record, &nuc_ends),
);
(msp_ends, msp_starts) = (
positions_on_complimented_sequence(record, &msp_starts),
positions_on_complimented_sequence(record, &msp_ends),
);
nuc_length = nuc_starts
.iter()
.zip(nuc_ends.iter())
.map(|(&x, &y)| y - x)
.collect::<Vec<_>>();
msp_length = msp_starts
.iter()
.zip(msp_ends.iter())
.map(|(&x, &y)| y - x)
.collect::<Vec<_>>();
}
let ref_nuc = get_closest_reference_range(&nuc_starts, &nuc_length, record);
let ref_msp = get_closest_reference_range(&msp_starts, &msp_length, record);
assert_eq!(ref_nuc.len(), nuc_starts.len());
assert_eq!(ref_msp.len(), msp_starts.len());
let ec = if let Ok(Aux::Float(f)) = record.aux(b"ec") {
log::trace!("{f}");
f
} else {
0.0
};
FiberseqData {
record: record.clone(),
nuc: nuc_starts
.iter()
.cloned()
.zip(nuc_length.iter().cloned())
.collect(),
msp: msp_starts
.iter()
.cloned()
.zip(msp_length.iter().cloned())
.collect(),
ref_nuc,
ref_msp,
base_mods: BaseMods::new(record, min_ml_score),
ec,
}
}
pub fn from_records(records: &Vec<bam::Record>, min_ml_score: u8) -> Vec<Self> {
records
.par_iter()
.map(|x| FiberseqData::new(x, min_ml_score))
.collect::<Vec<_>>()
}
pub fn get_nuc(&self, reference: bool, get_starts: bool) -> Vec<i64> {
let (starts, lengths): (Vec<_>, Vec<_>) = if reference {
self.ref_nuc.iter().map(|(a, b)| (a, b)).unzip()
} else {
self.nuc.iter().map(|(a, b)| (a, b)).unzip()
};
if get_starts {
starts
} else {
lengths
}
}
pub fn get_msp(&self, reference: bool, get_starts: bool) -> Vec<i64> {
let (starts, lengths): (Vec<_>, Vec<_>) = if reference {
self.ref_msp.iter().map(|(a, b)| (a, b)).unzip()
} else {
self.msp.iter().map(|(a, b)| (a, b)).unzip()
};
if get_starts {
starts
} else {
lengths
}
}
pub fn write_msp(&self, reference: bool, head_view: &HeaderView) -> String {
let starts = self.get_msp(reference, true);
let lengths = self.get_msp(reference, false);
if starts.is_empty() {
return "".to_string();
}
self.to_bed12(reference, &starts, &lengths, head_view)
}
pub fn write_nuc(&self, reference: bool, head_view: &HeaderView) -> String {
let starts = self.get_nuc(reference, true);
let lengths = self.get_nuc(reference, false);
if starts.is_empty() {
return "".to_string();
}
self.to_bed12(reference, &starts, &lengths, head_view)
}
pub fn write_m6a(&self, reference: bool, head_view: &HeaderView) -> String {
let starts = self.base_mods.m6a_positions(reference);
if starts.is_empty() {
return "".to_string();
}
let lengths = vec![1; starts.len()];
self.to_bed12(reference, &starts, &lengths, head_view)
}
pub fn write_cpg(&self, reference: bool, head_view: &HeaderView) -> String {
let starts = self.base_mods.cpg_positions(reference);
if starts.is_empty() {
return "".to_string();
}
let lengths = vec![1; starts.len()];
self.to_bed12(reference, &starts, &lengths, head_view)
}
pub fn get_rq(&self) -> Option<f32> {
if let Ok(Aux::Float(f)) = self.record.aux(b"rq") {
Some(f)
} else {
None
}
}
pub fn all_header(simplify: bool, quality: bool) -> String {
let mut x = format!(
"#{}\t{}\t{}\t{}\t{}\t{}\t{}\t",
"ct", "st", "en", "fiber", "score", "strand", "fiber_length",
);
if !simplify {
x.push_str("fiber_sequence\t")
}
if quality {
x.push_str("fiber_qual\t")
}
x.push_str(&format!(
"{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\n",
"ec",
"rq",
"total_AT_bp",
"total_m6a_bp",
"total_nuc_bp",
"total_msp_bp",
"total_5mC_bp",
"nuc_starts",
"nuc_lengths",
"ref_nuc_starts",
"ref_nuc_lengths",
"msp_starts",
"msp_lengths",
"ref_msp_starts",
"ref_msp_lengths",
"m6a",
"ref_m6a",
"m6a_qual",
"5mC",
"ref_5mC",
));
x
}
pub fn write_all(&self, head_view: &HeaderView, simplify: bool, quality: bool) -> String {
let name = std::str::from_utf8(self.record.qname()).unwrap();
let score = self.ec.round() as i64;
let q_len = self.record.seq_len() as i64;
let rq = match self.get_rq() {
Some(x) => format!("{}", x),
None => ".".to_string(),
};
let ct;
let start;
let end;
let strand;
if self.record.is_unmapped() {
ct = ".";
start = 0;
end = 0;
strand = '.';
} else {
ct = std::str::from_utf8(head_view.tid2name(self.record.tid() as u32)).unwrap();
start = self.record.reference_start();
end = self.record.reference_end();
strand = if self.record.is_reverse() { '-' } else { '+' };
}
let nuc_starts = self.get_nuc(false, true);
let nuc_lengths = self.get_nuc(false, false);
let ref_nuc_starts = self.get_nuc(true, true);
let ref_nuc_lengths = self.get_nuc(true, false);
let msp_starts = self.get_msp(false, true);
let msp_lengths = self.get_msp(false, false);
let ref_msp_starts = self.get_msp(true, true);
let ref_msp_lengths = self.get_msp(true, false);
let at_count = self
.record
.seq()
.as_bytes()
.iter()
.filter(|&x| *x == b'A' || *x == b'T')
.count() as i64;
let (m6a, _m6a_qual) = self.base_mods.m6a(false);
let m6a_count = m6a.len();
let (ref_m6a, m6a_qual) = self.base_mods.m6a(true);
let m6a_qual: Vec<i64> = m6a_qual.into_iter().map(|a| a as i64).collect();
let cpg = self.base_mods.cpg_positions(false);
let cpg_count = cpg.len();
let ref_cpg = self.base_mods.cpg_positions(true);
let mut rtn = String::with_capacity(0);
rtn.write_fmt(format_args!(
"{}\t{}\t{}\t{}\t{}\t{}\t{}\t",
ct, start, end, name, score, strand, q_len
))
.unwrap();
if !simplify {
rtn.write_fmt(format_args!(
"{}\t",
String::from_utf8_lossy(&self.record.seq().as_bytes()),
))
.unwrap();
}
if quality {
rtn.write_fmt(format_args!(
"{}\t",
String::from_utf8_lossy(
&self
.record
.qual()
.iter()
.map(|x| x + 33)
.collect::<Vec<u8>>()
),
))
.unwrap();
}
let total_nuc_bp = nuc_lengths.iter().sum::<i64>();
let total_msp_bp = msp_lengths.iter().sum::<i64>();
rtn.write_fmt(format_args!(
"{}\t{}\t{}\t{}\t{}\t{}\t{}\t",
self.ec, rq, at_count, m6a_count, total_nuc_bp, total_msp_bp, cpg_count
))
.unwrap();
for vec in &[
&nuc_starts,
&nuc_lengths,
&ref_nuc_starts,
&ref_nuc_lengths,
&msp_starts,
&msp_lengths,
&ref_msp_starts,
&ref_msp_lengths,
&m6a,
&ref_m6a,
&m6a_qual,
&cpg,
&ref_cpg,
] {
if vec.is_empty() {
rtn.push('.');
rtn.push('\t');
} else {
let z: String = vec.iter().map(|&x| x.to_string() + ",").collect();
rtn.write_fmt(format_args!("{}\t", z)).unwrap();
}
}
let len = rtn.len();
rtn.replace_range(len - 1..len, "\n");
rtn
}
pub fn to_bed12(
&self,
reference: bool,
starts: &[i64],
lengths: &[i64],
head_view: &HeaderView,
) -> String {
if self.record.is_unmapped() && reference {
return "".to_string();
}
let ct;
let start;
let end;
let name = std::str::from_utf8(self.record.qname()).unwrap();
let mut rtn: String = String::with_capacity(0);
if reference {
ct = std::str::from_utf8(head_view.tid2name(self.record.tid() as u32)).unwrap();
start = self.record.reference_start();
end = self.record.reference_end();
} else {
ct = name;
start = 0;
end = self.record.seq_len() as i64;
}
let score = self.ec.round() as i64;
let strand = if self.record.is_reverse() { '-' } else { '+' };
let color = "126,126,126";
let (filtered_starts, filtered_lengths): (Vec<i64>, Vec<i64>) = starts
.iter()
.zip(lengths.iter())
.filter(|(&st, &ln)| st >= 0 && ln >= 0)
.unzip();
let b_ct = filtered_starts.len() + 2;
let b_ln: String = filtered_lengths
.iter()
.map(|&ln| ln.to_string() + ",")
.collect();
let b_st: String = filtered_starts
.iter()
.map(|&st| (st - start).to_string() + ",")
.collect();
assert_eq!(filtered_lengths.len(), filtered_starts.len());
rtn.push_str(ct);
rtn.push('\t');
rtn.push_str(&start.to_string());
rtn.push('\t');
rtn.push_str(&end.to_string());
rtn.push('\t');
rtn.push_str(name);
rtn.push('\t');
rtn.push_str(&score.to_string());
rtn.push('\t');
rtn.push(strand);
rtn.push('\t');
rtn.push_str(&start.to_string());
rtn.push('\t');
rtn.push_str(&end.to_string());
rtn.push('\t');
rtn.push_str(color);
rtn.push('\t');
rtn.push_str(&b_ct.to_string());
rtn.push_str("\t0,"); rtn.push_str(&b_ln);
rtn.push_str("1\t0,"); rtn.push_str(&b_st);
write!(&mut rtn, "{}", format_args!("{}\n", end - start - 1)).unwrap();
rtn
}
}
#[allow(clippy::too_many_arguments)]
pub fn process_bam_chunk(
records: &Vec<bam::Record>,
so_far: usize,
out_files: &mut FiberOut,
head_view: &HeaderView,
) {
let start = Instant::now();
let mut fiber_data = FiberseqData::from_records(records, out_files.min_ml_score);
match &mut out_files.m6a {
Some(m6a) => {
let out: Vec<String> = fiber_data
.iter_mut()
.map(|r| r.write_m6a(out_files.reference, head_view))
.collect();
for line in out {
write_to_file(&line, m6a);
}
}
None => {}
}
match &mut out_files.cpg {
Some(cpg) => {
let out: Vec<String> = fiber_data
.iter_mut()
.map(|r| r.write_cpg(out_files.reference, head_view))
.collect();
for line in out {
write_to_file(&line, cpg);
}
}
None => {}
}
match &mut out_files.msp {
Some(msp) => {
let out: Vec<String> = fiber_data
.iter_mut()
.map(|r| r.write_msp(out_files.reference, head_view))
.collect();
for line in out {
write_to_file(&line, msp);
}
}
None => {}
}
match &mut out_files.nuc {
Some(nuc) => {
let out: Vec<String> = fiber_data
.iter_mut()
.map(|r| r.write_nuc(out_files.reference, head_view))
.collect();
for line in out {
write_to_file(&line, nuc);
}
}
None => {}
}
match &mut out_files.all {
Some(all) => {
let out: Vec<String> = fiber_data
.iter_mut()
.map(|r| r.write_all(head_view, out_files.simplify, out_files.quality))
.collect();
for line in out {
write_to_file(&line, all);
}
}
None => {}
}
let duration = start.elapsed().as_secs_f64();
log::info!(
"Processing {}, {} reads done so far.",
format!("{:.2?} reads/s", records.len() as f64 / duration)
.bright_cyan()
.bold(),
format!("{:}", so_far + records.len())
.bright_magenta()
.bold()
);
}
pub fn extract_contained(bam: &mut bam::Reader, mut out_files: FiberOut) {
let header = bam::Header::from_template(bam.header());
let head_view = bam::HeaderView::from_header(&header);
match &mut out_files.all {
Some(all) => {
write!(
all,
"{}",
FiberseqData::all_header(out_files.simplify, out_files.quality)
)
.unwrap();
}
None => {}
}
let bin_size = current_num_threads() * 500;
let mut cur_count = 0;
let mut cur_vec = vec![];
let mut proccesed_reads = 0;
for r in bam.records() {
let record = r.unwrap();
cur_vec.push(record);
cur_count += 1;
if cur_count == bin_size {
process_bam_chunk(&cur_vec, proccesed_reads, &mut out_files, &head_view);
proccesed_reads += cur_vec.len();
cur_vec.clear();
cur_count = 0;
}
}
process_bam_chunk(&cur_vec, proccesed_reads, &mut out_files, &head_view);
}