Thomas 1 year ago
commit
2af90cd37b
5 changed files with 4187 additions and 0 deletions
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  4. 104 0
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+ 19 - 0
Cargo.toml

@@ -0,0 +1,19 @@
+[package]
+name = "pandora_lib_assembler"
+version = "0.1.0"
+edition = "2021"
+
+[dependencies]
+anyhow = "1.0.86"
+duct = "0.13.7"
+noodles-fasta = "0.41.0"
+noodles-fastq = "0.13.0"
+rust-htslib = "0.46.0"
+uuid = { version = "1.10.0", features = ["v4"] }
+pandora_lib_blastn = { git = "https://git.t0m4.fr/Thomas/pandora_lib_blastn.git" }
+nom = "7.1.3"
+petgraph = "0.6.5"
+bam = "0.1.4"
+log = "0.4.22"
+flate2 = "1.0.30"
+

+ 104 - 0
src/flye.rs

@@ -0,0 +1,104 @@
+use log::{info, warn};
+use std::{
+    fs::{self, File}, io::{BufRead, BufReader}, path::Path, process::{Command, Stdio}
+};
+
+pub fn run_flye(fasta_path: &str, tmp_dir: &str, _size: &str) {
+    info!("Running Flye in {tmp_dir}");
+    let flye_bin = "/data/tools/Flye/bin/flye";
+    let mut cmd = Command::new(flye_bin)
+        .arg("--out-dir")
+        .arg(tmp_dir)
+        .arg("--nano-hq")
+        .arg(fasta_path)
+        .arg("--threads")
+        .arg("12")
+        .arg("--min-overlap")
+        .arg("1000")
+        .stderr(Stdio::piped())
+        .spawn()
+        .expect("Flye failed to start");
+
+    let stderr = cmd.stderr.take().unwrap();
+    let reader = BufReader::new(stderr);
+    reader
+        .lines()
+        .map_while(Result::ok)
+        .filter(|line| line.contains("ERROR"))
+        .for_each(|line| warn!("[FLYE] {line}"));
+
+    cmd.wait().unwrap();
+    cmd.kill().unwrap();
+}
+
+pub fn read_flye_coverage(path: &str, min_cov: i32, contig_name: &str) -> (usize, usize) {
+    use std::io::Read;
+    let mut reader = File::open(path).map(flate2::read::GzDecoder::new).unwrap();
+    let mut buf = Vec::new();
+    reader.read_to_end(&mut buf).unwrap();
+
+    let mut line_acc = Vec::new();
+    let mut start = None;
+    let mut end = None;
+    let mut last_end = 0;
+    for b in buf.iter() {
+        match b {
+            b'\n' => {
+                let s = String::from_utf8(line_acc.clone()).unwrap();
+                line_acc.clear();
+                if !s.starts_with(&format!("{contig_name}\t")) {
+                    continue;
+                }
+                let s = s.split('\t').collect::<Vec<&str>>();
+                let cov: i32 = s.get(3).unwrap().parse().unwrap();
+                if start.is_none() && cov >= min_cov {
+                    let st: i32 = s.get(1).unwrap().parse().unwrap();
+                    start = Some(st);
+                } else if end.is_none() && start.is_some() && cov < min_cov {
+                    let en: i32 = s.get(1).unwrap().parse().unwrap();
+                    end = Some(en);
+                    break;
+                }
+                last_end = s.get(2).unwrap().parse().unwrap();
+            }
+            _ => line_acc.push(*b),
+        }
+    }
+    (start.unwrap() as usize, end.unwrap_or(last_end) as usize)
+}
+
+pub fn assemble_flye(reads_path: &str) -> anyhow::Result<Option<Vec<String>>> {
+    let min_cov = 2;
+
+    let tmp_dir = format!("/tmp/ass_{}", uuid::Uuid::new_v4());
+    fs::create_dir(tmp_dir.clone()).unwrap();
+
+    run_flye(reads_path, &tmp_dir, "10M");
+
+    let contigs_path = format!("{tmp_dir}/assembly.fasta");
+    let mut res = None;
+
+    if Path::new(&contigs_path).exists() {
+        let assembly_path = format!("{tmp_dir}/assembly.fasta");
+        let flye_cov_path = format!("{tmp_dir}/40-polishing/base_coverage.bed.gz");
+
+        let mut reader = File::open(assembly_path)
+            .map(BufReader::new)
+            .map(noodles_fasta::Reader::new)?;
+        let mut contigs = Vec::new();
+        for result in reader.records() {
+            let record = result?;
+            let contig_name = String::from_utf8(record.name().to_vec()).unwrap();
+            let (s, e) = read_flye_coverage(&flye_cov_path, min_cov, &contig_name);
+            let seq = record.sequence().as_ref();
+            let seq = String::from_utf8(seq.to_vec()).unwrap();
+            let seq: String = seq[s..e].into();
+            contigs.push(seq.clone());
+        }
+        res = Some(contigs);
+    }
+    fs::remove_dir_all(tmp_dir)?;
+    Ok(res)
+}
+
+

+ 485 - 0
src/lib.rs

@@ -0,0 +1,485 @@
+pub mod flye;
+
+use bam::{header::HeaderEntry, RecordWriter};
+use pandora_lib_blastn::BlastResult;
+use petgraph::{
+    algo::dijkstra,
+    data::{Build, DataMap},
+    dot::Dot,
+    graph::NodeIndex,
+    stable_graph::StableUnGraph,
+    visit::{EdgeRef, IntoNeighbors, NodeIndexable},
+};
+use rust_htslib::bam::{Read, Reader, Record};
+use std::{
+    collections::{HashMap, HashSet, VecDeque},
+    fs::{self, File},
+    io::{BufReader, Cursor, Write},
+    path::{Path, PathBuf},
+};
+use uuid::Uuid;
+
+pub fn read_bam(path: &str) -> anyhow::Result<Vec<Record>> {
+    let mut res = Vec::new();
+    let mut bam = Reader::from_path(Path::new(path))?;
+
+    // Iterate over all records
+    for result in bam.records() {
+        let record = result?;
+        res.push(record);
+    }
+
+    Ok(res)
+}
+
+fn write_fastqs(dir_path: &str, records: &Vec<Record>) -> anyhow::Result<Vec<String>> {
+    let mut paths = Vec::new();
+    for record in records {
+        let qname = String::from_utf8(record.qname().to_vec())?;
+        let qseq = record.seq().as_bytes();
+        let qqual = String::from_utf8(record.qual().to_vec())?;
+        assert_eq!(qseq.len(), qqual.len());
+        let fastq_record = noodles_fasta::Record::new(
+            noodles_fasta::record::Definition::new(qname.clone(), None),
+            qseq.into(),
+        );
+        let mut writer = noodles_fasta::io::Writer::new(Vec::new());
+        writer.write_record(&fastq_record)?;
+        let file_path = format!("{dir_path}/{qname}.fasta");
+        let mut file = File::create(&file_path)?;
+        file.write_all(writer.get_ref())?;
+        paths.push(file_path);
+    }
+    Ok(paths)
+}
+
+pub fn lastz_two_by_two(records: &[String], best: bool) -> anyhow::Result<Vec<BlastResult>> {
+    let lastz_bin = "lastz";
+    let mut all: Vec<BlastResult> = Vec::new();
+    for i in 0..records.len() {
+        for j in (i + 1)..records.len() {
+            let record_a = &records[i];
+            let record_b = &records[j];
+            let out = duct::cmd!(lastz_bin, "--format=blastn", record_a, record_b).read()?;
+            let mut results: Vec<pandora_lib_blastn::BlastResult> = out
+                .lines()
+                .filter(|s| !s.starts_with('#'))
+                .map(|s| s.to_string())
+                .filter_map(|s| BlastResult::try_from(s).ok())
+                .collect();
+            if !results.is_empty() {
+                if best {
+                    results.sort_by_key(|r| r.alignment_length);
+                    all.push(results.last().unwrap().clone())
+                } else {
+                    all.extend(results.into_iter());
+                }
+            }
+        }
+    }
+
+    Ok(all)
+}
+
+pub fn lastz_bam(query_path: &str, subject_path: &str) -> anyhow::Result<Vec<bam::Record>> {
+    let lastz_bin = "lastz";
+    let pipe = format!("{lastz_bin} --format=sam {query_path} {subject_path} | samtools view -b");
+    let out = duct::cmd!("bash", "-c", pipe).reader()?;
+    let reader = bam::BamReader::from_stream(out, 4)?;
+    Ok(reader.into_iter().filter_map(anyhow::Result::ok).collect())
+}
+
+pub fn lastz_remap(bam_path: &str, ref_fa: &str, ref_len: u32, new_bam: &str) -> anyhow::Result<()> {
+    let tmp = "/tmp";
+    let tmp_dir = format!("{tmp}/{}", Uuid::new_v4());
+    fs::create_dir(&tmp_dir)?;
+
+    let mut records = Vec::new();
+    let reader = bam::BamReader::from_path(bam_path, 4).unwrap();
+    for record in reader {
+        let record = record?;
+        let query_name = String::from_utf8(record.name().to_vec())?;
+        let query_path = format!("{}/{}.fa", &tmp_dir, query_name);
+        println!("writing fasta {}", &query_path);
+        let query_seq = String::from_utf8(record.sequence().to_vec())?;
+
+        write_fasta(&query_path, &vec![(query_name.clone(), query_seq)]);
+        let mut res = lastz_bam(&query_path, ref_fa)?;
+        // take best length
+        res.sort_by_cached_key(|r| r.aligned_query_end() - r.aligned_query_start());
+        let mut r = res.last().unwrap().clone();
+        r.set_name(query_name.as_bytes().to_vec().clone());
+        records.push(r);
+    }
+    records.sort_by_cached_key(|b| b.start());
+
+    let contig_name = PathBuf::from(&ref_fa).file_stem().unwrap().to_string_lossy().to_string();
+    let mut header = bam::header::Header::new();
+    let mut header_line = HeaderEntry::header_line("1.6".to_string());
+    header_line.push(b"SO", "Coordinate".to_string());
+    header.push_entry(header_line).unwrap();
+    header.push_entry(HeaderEntry::ref_sequence(contig_name, ref_len +10_000)).unwrap();
+
+    let mut bam_writer = bam::bam_writer::BamWriterBuilder::new();
+    let mut w = bam_writer.from_path(new_bam, header)?;
+    for record in records.iter() {
+        w.write(record)?;
+    }
+    w.finish()?;
+    fs::remove_dir_all(tmp_dir)?;
+
+    duct::cmd!("samtools", "index", "-@", "10", new_bam).run()?;
+    Ok(())
+}
+
+pub fn write_fasta(fasta_path: &str, d: &Vec<(String, String)>) {
+    let file = File::create(fasta_path).unwrap();
+    let mut writer = noodles_fasta::writer::Builder::default().build_with_writer(file);
+    let mut passed = Vec::new();
+    for (name, sequence) in d {
+        let name = name.to_string();
+        if sequence.is_empty() {
+            continue;
+        }
+        if passed.contains(&name) {
+            continue;
+        }
+        passed.push(name.clone());
+        let record = noodles_fasta::Record::new(
+            noodles_fasta::record::Definition::new(name.as_str(), None),
+            noodles_fasta::record::Sequence::from(sequence.as_bytes().to_vec()),
+        );
+        writer.write_record(&record).unwrap();
+    }
+}
+
+pub fn make_graph(d: Vec<BlastResult>, records: &Vec<Record>) {
+    let mut g: StableUnGraph<String, Option<BlastResult>> = StableUnGraph::default();
+    let mut name_to_id: HashMap<String, NodeIndex> = HashMap::new();
+
+    for blast_result in d {
+        let nid_a = if let Some(nid_a) = name_to_id.get(&blast_result.query_id) {
+            *nid_a
+        } else {
+            let k = blast_result.query_id.clone();
+            let nid_a = g.add_node(k.clone());
+            name_to_id.insert(k, nid_a);
+            nid_a
+        };
+        let nid_b = if let Some(nid_b) = name_to_id.get(&blast_result.subject_id) {
+            *nid_b
+        } else {
+            let k = blast_result.subject_id.clone();
+            let nid_b = g.add_node(k.clone());
+            name_to_id.insert(k, nid_b);
+            nid_b
+        };
+        g.add_edge(nid_a, nid_b, Some(blast_result));
+    }
+
+    println!(
+        "{:?}",
+        Dot::with_config(&g, &[petgraph::dot::Config::EdgeNoLabel])
+    );
+    // let order = dijkstra(&g, start_node, None, |_| 0);
+    // println!("{order:#?}");
+
+    // begin with start node
+    let mut n_neigh: Vec<(usize, NodeIndex)> = g
+        .node_indices()
+        .map(|nid| (g.neighbors(nid).count(), nid))
+        .collect();
+    n_neigh.sort_by_key(|v| v.0);
+    let start_node = n_neigh.last().unwrap().1;
+    let start_name = g.node_weight(start_node).unwrap().to_string();
+
+    let neighbors = g.neighbors(start_node);
+    let mut neighbors_blast = Vec::new();
+    for neighbor in neighbors {
+        let e: Vec<BlastResult> = g
+            .edges_connecting(start_node, neighbor)
+            .map(|e| e.weight().clone().unwrap())
+            .collect();
+        neighbors_blast.extend(e);
+    }
+    let mut neighbors_blast: Vec<(BlastResult, usize)> = neighbors_blast
+        .iter()
+        .map(|b| {
+            if b.subject_id != start_name {
+                b.swap_query_subject()
+            } else {
+                b.clone()
+            }
+        })
+        .map(|b| (b.clone(), get_seq_len(records, &b.query_id)))
+        .collect();
+    neighbors_blast.sort_by_key(|b| b.0.s_end);
+
+    let start_len = get_seq_len(records, &start_name);
+    println!("{start_len}");
+    println!("{neighbors_blast:#?}");
+}
+
+pub fn get_seq_len(records: &[Record], id: &str) -> usize {
+    let lens: Vec<usize> = records
+        .iter()
+        .filter(|r| String::from_utf8(r.qname().to_vec()).unwrap() == id)
+        .map(|r| r.seq_len())
+        .collect();
+    lens[0]
+}
+
+// pub fn lastz_two_by_two_bam(records: &[String]) -> anyhow::Result<Vec<BlastResult>> {
+//     let lastz_bin = "lastz";
+//     let mut all: Vec<bam::Record> = Vec::new();
+//     for i in 0..records.len() {
+//         for j in (i + 1)..records.len() {
+//             let pipe = format!(
+//                 "{lastz_bin} --format=sam {} {} | samtools view -b",
+//                 &records[i], &records[j]
+//             );
+//             println!("{pipe}");
+//             let child = duct::cmd!("bash", "-c", pipe).reader()?;
+//             println!("mmm");
+//             let reader = bam::BamReader::from_stream(child, 3).unwrap();
+//             reader.header();
+//             println!("lll");
+//             let mut records = Vec::new();
+//             for r in reader {
+//                 let record = r?;
+//                 records.push(record);
+//             }
+//             if !records.is_empty() {
+//                     records.sort_by_key(|r| r.template_len());
+//                     all.push(records.last().unwrap().clone())
+//             }
+//
+//             // let bam_reader = rust_htslib::bam::Reader::from_reader(buf_reader);
+//         }
+//     }
+//
+//     Ok(all)
+// }
+
+pub fn order_sequences_with_positions(blast_results: &[BlastResult]) -> Vec<(String, usize)> {
+    let mut graph: HashMap<String, HashSet<String>> = HashMap::new();
+    let mut in_degree: HashMap<String, usize> = HashMap::new();
+    let mut all_sequences: HashSet<String> = HashSet::new();
+
+    // Build the graph
+    for result in blast_results {
+        all_sequences.insert(result.query_id.clone());
+        all_sequences.insert(result.subject_id.clone());
+
+        if result.q_start < result.s_start {
+            graph
+                .entry(result.query_id.clone())
+                .or_insert_with(HashSet::new)
+                .insert(result.subject_id.clone());
+            *in_degree.entry(result.subject_id.clone()).or_insert(0) += 1;
+        } else {
+            graph
+                .entry(result.subject_id.clone())
+                .or_insert_with(HashSet::new)
+                .insert(result.query_id.clone());
+            *in_degree.entry(result.query_id.clone()).or_insert(0) += 1;
+        }
+    }
+
+    // Topological sort
+    let mut queue: VecDeque<String> = all_sequences
+        .iter()
+        .filter(|&seq| !in_degree.contains_key(seq))
+        .cloned()
+        .collect();
+    let mut ordered_sequences: Vec<String> = Vec::new();
+
+    while let Some(seq) = queue.pop_front() {
+        ordered_sequences.push(seq.clone());
+
+        if let Some(neighbors) = graph.get(&seq) {
+            for neighbor in neighbors {
+                if let Some(degree) = in_degree.get_mut(neighbor) {
+                    *degree -= 1;
+                    if *degree == 0 {
+                        queue.push_back(neighbor.clone());
+                    }
+                }
+            }
+        }
+    }
+
+    // Handle any remaining sequences (in case of cycles)
+    for seq in all_sequences {
+        if !ordered_sequences.contains(&seq) {
+            ordered_sequences.push(seq);
+        }
+    }
+
+    // Calculate absolute positions
+    let mut positions: HashMap<String, usize> = HashMap::new();
+    let mut current_position = 0;
+
+    for seq in &ordered_sequences {
+        positions.insert(seq.clone(), current_position);
+        current_position += 1; // Increment position for each sequence
+    }
+
+    let mut res: Vec<_> = ordered_sequences
+        .into_iter()
+        .map(|seq| (seq.clone(), *positions.get(&seq).unwrap()))
+        .collect();
+    res.sort_by(|a, b| a.1.cmp(&b.1));
+    res
+}
+
+// fn order_sequences_with_positions_bam(records: &Vec<bam::Record>, header: &Header) -> anyhow::Result<Vec<(String, usize)>> {
+//     let mut graph: HashMap<String, HashSet<String>> = HashMap::new();
+//     let mut in_degree: HashMap<String, usize> = HashMap::new();
+//     let mut all_sequences: HashSet<String> = HashSet::new();
+//
+//     for record in records {
+//         let query_id = String::from_utf8(record.name().to_vec());
+//         let subject_id = record.ref_id().clone();
+//         let q_start = record.aligned_query_start();
+//         let s_start = record.start();
+//
+//         all_sequences.insert(query_id.clone());
+//         all_sequences.insert(subject_id.clone());
+//
+//         if q_start < s_start {
+//             graph.entry(query_id.clone())
+//                 .or_insert_with(HashSet::new())
+//                 .insert(subject_id.clone());
+//             *in_degree.entry(subject_id.clone()).or_insert(0) += 1;
+//         } else {
+//             graph.entry(subject_id.clone())
+//                 .or_insert_with(HashSet::new())
+//                 .insert(query_id.clone());
+//             *in_degree.entry(query_id.clone()).or_insert(0) += 1;
+//         }
+//     }
+//
+//     let mut queue: VecDeque<String> = all_sequences.iter()
+//         .filter(|&seq| !in_degree.contains_key(seq))
+//         .cloned()
+//         .collect();
+//
+//     let mut ordered_sequences: Vec<String> = Vec::new();
+//
+//     while let Some(seq) = queue.pop_front() {
+//         ordered_sequences.push(seq.clone());
+//
+//         if let Some(neighbors) = graph.get(&seq) {
+//             for neighbor in neighbors {
+//                 if let Some(degree) = in_degree.get_mut(neighbor) {
+//                     *degree -= 1;
+//                     if *degree == 0 {
+//                         queue.push_back(neighbor.clone());
+//                     }
+//                 }
+//             }
+//         }
+//     }
+//
+//     for seq in all_sequences {
+//         if !ordered_sequences.contains(&seq) {
+//             ordered_sequences.push(seq.clone());
+//         }
+//     }
+//
+//     let mut positions: HashMap<String, usize> = HashMap::new();
+//     let mut current_position = 0;
+//
+//     for seq in &ordered_sequences {
+//         positions.insert(seq.clone(), current_position);
+//         current_position += 1;
+//     }
+//
+//     ordered_sequences.into_iter()
+//         .map(|seq| (seq.clone(), *positions.get(&seq).unwrap()))
+//         .collect()
+// }
+
+pub fn fai(path: &str) -> Result<std::process::Output, std::io::Error> {
+    duct::cmd!("samtools", "faidx", path).run()
+}
+
+
+#[cfg(test)]
+mod tests {
+    use std::fs;
+
+    use uuid::Uuid;
+
+    use self::flye::assemble_flye;
+
+    use super::*;
+
+    #[test]
+    fn it_works() -> anyhow::Result<()> {
+        let dir = "/tmp";
+        let records  = read_bam("/home/prom/Documents/Programmes/pandora_lib_pileup/44d3d2a9-ad53-45f7-9a1a-2e1e229377e4.bam")?;
+        println!("{} rec", records.len());
+
+        let tmp_dir = format!("{dir}/ass_{}", Uuid::new_v4());
+        println!("{tmp_dir}");
+        fs::create_dir(&tmp_dir)?;
+        let paths = write_fastqs(&tmp_dir, &records)?;
+        println!("kkkk");
+        let blast_results = lastz_two_by_two(&paths, true)?;
+        // println!("{blast_results:#?}");
+        fs::remove_dir_all(tmp_dir)?;
+        make_graph(blast_results, &records);
+        // let res = order_sequences_with_positions(&blast_results);
+        // println!("{res:#?}");
+        // let leftmost = res.first().unwrap().0.clone();
+        // let mut on_lm: Vec<BlastResult> = blast_results
+        //     .iter()
+        //     .filter(|b| b.subject_id == leftmost || b.query_id == leftmost)
+        //     .map(|b| {
+        //         if b.query_id == leftmost {
+        //             b.swap_query_subject()
+        //         } else {
+        //             b.clone()
+        //         }
+        //     })
+        //     .collect();
+        // on_lm.sort_by(|a, b| a.s_start.cmp(&b.s_start));
+        // println!("{on_lm:#?}");
+        Ok(())
+    }
+
+    #[test]
+    fn flye() -> anyhow::Result<()> {
+        let id = "BECERRA";
+        let locus_id = "6d51d94e-b951-48ef-9cef-63c10f235ebe";
+        let res_dir = "/data/longreads_basic_pipe";
+        let asm_dir = format!("{res_dir}/{id}/diag/asm_contigs");
+        let path = format!("{asm_dir}/{locus_id}.fasta");
+        let bam_path = format!("{asm_dir}/{locus_id}.bam");
+        let new_bam = format!("{asm_dir}/{locus_id}_contig.bam");
+
+        let contigs = assemble_flye(&path)?.unwrap();
+        if contigs.len() == 1 {
+            let contig = contigs[0].to_string();
+            let fasta_path = format!("{asm_dir}/{locus_id}_flye.fa");
+            let bed_path = format!("{asm_dir}/{locus_id}_flye.bed");
+            write_fasta(&fasta_path, &vec![(format!("{}_flye", locus_id), contig.clone())]);
+            let bed = pandora_lib_blastn::Blast::init(&fasta_path)?
+                .run()?
+                .keep_best()
+                .to_bed()?;
+            let mut f = File::create(bed_path)?;
+            f.write_all(bed.as_bytes())?;
+            fai(&fasta_path)?;
+            lastz_remap(&bam_path, &fasta_path, contig.len() as u32, &new_bam)?;
+
+        } else {
+            for (i, contig) in contigs.iter().enumerate() {
+                println!("{contig}");
+            }
+        }
+        Ok(())
+    }
+}