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# 线程数 MM2_THREADS=36 ST_THREADS=36 # 软件绝对路径（minim...

创建于：2025年12月9日

使用 GPT-5.1 Thinking 回答 Chat01

问题

线程数

MM2_THREADS=36
ST_THREADS=36

软件绝对路径（minimap2 在环境里，直接调用）

SAMTOOLS="/public/Zhaijx/wangz/Condaenvir/envs/samtools/bin/samtools"
SEQKIT="/public/Zhaijx/wangz/Condaenvir/envs/seqkit/bin/seqkit"

参考基因组（Col-CC-V2）

REF="/DataZhai/wangzh/genome/Col-CC-V2/Col-CC-V2.fasta"

原始输入 FASTQ（如有更新，只改这行即可）

IN_FASTQ="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/fastq/Col-cotyledon-dor-pass-fail-sup.fastq"

输出目录

OUTDIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2"
mkdir -p "${OUTDIR}"

sample 名，用来拼接输出文件前缀

SAMPLE="Col-coty-dorado"
BASE="OUTDIR/{OUTDIR}/{SAMPLE}-Col-CC-V2"

###############################################################################

Round 1：minimap2 map-ont 对原始 FASTQ

###############################################################################
R1_PREFIX="BASE−r1−mapont"R1SAM="{BASE}-r1-mapont" R1_SAM="{R1_PREFIX}.sam"
R1_BAM="R1PREFIX.bam"R1SORTEDBAM="{R1_PREFIX}.bam" R1_SORTED_BAM="{R1_PREFIX}-sorted.bam"
R1_UNMAPPED_BAM="R1PREFIX−sorted−unmapped.bam"R1UNMAPPEDIDS="{R1_PREFIX}-sorted-unmapped.bam" R1_UNMAPPED_IDS="{R1_PREFIX}-sorted.unmapped_read_ids.txt"
R1_UNMAPPED_IDS_CLEAN="R1PREFIX−sorted.unmappedreadids.clean.txt"R1UNMAPPEDFASTQ="{R1_PREFIX}-sorted.unmapped_read_ids.clean.txt" R1_UNMAPPED_FASTQ="{R1_PREFIX}.unmapped.fq.gz"

echo "[R1] minimap2 map-ont: INFASTQ"minimap2−xmap−ont−a−−MD−−cs=long −t{IN_FASTQ}" minimap2 -x map-ont -a --MD --cs=long \ -t {MM2_THREADS}
"REF""{REF}" "{IN_FASTQ}" > "${R1_SAM}"

echo "[R1] SAM -> BAM & sort"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -o "R1BAM""{R1_BAM}" "{R1_SAM}"
"SAMTOOLS"sort−@{SAMTOOLS}" sort -@ {ST_THREADS} -o "R1SORTEDBAM""{R1_SORTED_BAM}" "{R1_BAM}"
"SAMTOOLS"index−@{SAMTOOLS}" index -@ {ST_THREADS} "${R1_SORTED_BAM}"

echo "[R1] 提取未比对 reads 的 ID (flag 4)"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -f 4 "R1SORTEDBAM"−o"{R1_SORTED_BAM}" -o "{R1_UNMAPPED_BAM}"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} "{R1_UNMAPPED_BAM}" | awk '{print 1}' > "${R1_UNMAPPED_IDS}"

tr -d '\r' < "R1UNMAPPEDIDS"∣awk′length({R1_UNMAPPED_IDS}" | awk 'length(0)>0' | LC_ALL=C sort -u > "${R1_UNMAPPED_IDS_CLEAN}"

echo "[R1] 未比对 ID 行数：(wc−l<"(wc -l < "{R1_UNMAPPED_IDS_CLEAN}")"

echo "[R1] 使用 seqkit 从原始 FASTQ 抽取未比对 reads"

这里只提取 ID（QNAME），seqkit 默认按 ID 匹配，不用 -n

"SEQKIT"grep−j{SEQKIT}" grep -j {ST_THREADS} -f "R1UNMAPPEDIDSCLEAN""{R1_UNMAPPED_IDS_CLEAN}" "{IN_FASTQ}" -o "${R1_UNMAPPED_FASTQ}"

echo "[R1] R1_unmapped FASTQ 统计："
"SEQKIT"stats"{SEQKIT}" stats "{R1_UNMAPPED_FASTQ}"

R1 只删除原始 SAM 和未排序 BAM

echo "[R1] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "R1SAM""{R1_SAM}" "{R1_BAM}"

###############################################################################

Round 2：minimap2 asm20，对 R1 未比对 reads

###############################################################################
R2_PREFIX="BASE−r2−asm20"R2SAM="{BASE}-r2-asm20" R2_SAM="{R2_PREFIX}.sam"
R2_BAM="R2PREFIX.bam"R2SORTEDBAM="{R2_PREFIX}.bam" R2_SORTED_BAM="{R2_PREFIX}-sorted.bam"
R2_UNMAPPED_BAM="R2PREFIX−sorted−unmapped.bam"R2UNMAPPEDIDS="{R2_PREFIX}-sorted-unmapped.bam" R2_UNMAPPED_IDS="{R2_PREFIX}-sorted.unmapped_read_ids.txt"
R2_UNMAPPED_IDS_CLEAN="R2PREFIX−sorted.unmappedreadids.clean.txt"R2UNMAPPEDFASTQ="{R2_PREFIX}-sorted.unmapped_read_ids.clean.txt" R2_UNMAPPED_FASTQ="{R2_PREFIX}.unmapped.fq.gz"

echo "[R2] minimap2 asm20: R1UNMAPPEDFASTQ"minimap2−xasm20−a−−MD−−cs=long −t{R1_UNMAPPED_FASTQ}" minimap2 -x asm20 -a --MD --cs=long \ -t {MM2_THREADS}
"REF""{REF}" "{R1_UNMAPPED_FASTQ}" > "${R2_SAM}"

echo "[R2] SAM -> BAM & sort"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -o "R2BAM""{R2_BAM}" "{R2_SAM}"
"SAMTOOLS"sort−@{SAMTOOLS}" sort -@ {ST_THREADS} -o "R2SORTEDBAM""{R2_SORTED_BAM}" "{R2_BAM}"
"SAMTOOLS"index−@{SAMTOOLS}" index -@ {ST_THREADS} "${R2_SORTED_BAM}"

echo "[R2] 提取未比对 reads 的 ID (flag 4)"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -f 4 "R2SORTEDBAM"−o"{R2_SORTED_BAM}" -o "{R2_UNMAPPED_BAM}"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} "{R2_UNMAPPED_BAM}" | awk '{print 1}' > "${R2_UNMAPPED_IDS}"

tr -d '\r' < "R2UNMAPPEDIDS"∣awk′length({R2_UNMAPPED_IDS}" | awk 'length(0)>0' | LC_ALL=C sort -u > "${R2_UNMAPPED_IDS_CLEAN}"

echo "[R2] 未比对 ID 行数：(wc−l<"(wc -l < "{R2_UNMAPPED_IDS_CLEAN}")"

echo "[R2] 使用 seqkit 从 R1_unmapped FASTQ 抽取 R2 未比对 reads"
"SEQKIT"grep−j{SEQKIT}" grep -j {ST_THREADS} -f "R2UNMAPPEDIDSCLEAN""{R2_UNMAPPED_IDS_CLEAN}" "{R1_UNMAPPED_FASTQ}" -o "${R2_UNMAPPED_FASTQ}"

echo "[R2] R2_unmapped FASTQ 统计："
"SEQKIT"stats"{SEQKIT}" stats "{R2_UNMAPPED_FASTQ}"

R2 只删除原始 SAM 和未排序 BAM

echo "[R2] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "R2SAM""{R2_SAM}" "{R2_BAM}"

###############################################################################

Round 3：plan A（自定义 map-ont），对 R2 未比对 reads

###############################################################################
R3_PREFIX="BASE−r3−planA−mapont"R3SAM="{BASE}-r3-planA-mapont" R3_SAM="{R3_PREFIX}.sam"
R3_BAM="R3PREFIX.bam"R3SORTEDBAM="{R3_PREFIX}.bam" R3_SORTED_BAM="{R3_PREFIX}-sorted.bam"
R3_UNMAPPED_BAM="R3PREFIX−sorted−unmapped.bam"R3UNMAPPEDIDS="{R3_PREFIX}-sorted-unmapped.bam" R3_UNMAPPED_IDS="{R3_PREFIX}-sorted.unmapped_read_ids.txt"
R3_UNMAPPED_IDS_CLEAN="R3PREFIX−sorted.unmappedreadids.clean.txt"R3UNMAPPEDFASTQ="{R3_PREFIX}-sorted.unmapped_read_ids.clean.txt" R3_UNMAPPED_FASTQ="{R3_PREFIX}.unmapped.fq.gz" # 最终三轮都未比对上的 reads

echo "[R3][planA] minimap2 map-ont with tuned params: R2UNMAPPEDFASTQ"minimap2−xmap−ont−a−−MD−−cs=long −k13−w7 −r2k,60k−g8k −U40,700 −s70−z600,200 −N30−t{R2_UNMAPPED_FASTQ}" minimap2 -x map-ont -a --MD --cs=long \ -k13 -w7 \ -r2k,60k -g8k \ -U40,700 \ -s70 -z600,200 \ -N30 -t {MM2_THREADS}
"REF""{REF}" "{R2_UNMAPPED_FASTQ}" > "${R3_SAM}"

echo "[R3] SAM -> BAM & sort"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -o "R3BAM""{R3_BAM}" "{R3_SAM}"
"SAMTOOLS"sort−@{SAMTOOLS}" sort -@ {ST_THREADS} -o "R3SORTEDBAM""{R3_SORTED_BAM}" "{R3_BAM}"
"SAMTOOLS"index−@{SAMTOOLS}" index -@ {ST_THREADS} "${R3_SORTED_BAM}"

echo "[R3] 提取未比对 reads 的 ID (flag 4)"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} -b -f 4 "R3SORTEDBAM"−o"{R3_SORTED_BAM}" -o "{R3_UNMAPPED_BAM}"
"SAMTOOLS"view−@{SAMTOOLS}" view -@ {ST_THREADS} "{R3_UNMAPPED_BAM}" | awk '{print 1}' > "${R3_UNMAPPED_IDS}"

tr -d '\r' < "R3UNMAPPEDIDS"∣awk′length({R3_UNMAPPED_IDS}" | awk 'length(0)>0' | LC_ALL=C sort -u > "${R3_UNMAPPED_IDS_CLEAN}"

echo "[R3] 未比对 ID 行数：(wc−l<"(wc -l < "{R3_UNMAPPED_IDS_CLEAN}")"

echo "[R3] 使用 seqkit 从 R2_unmapped FASTQ 抽取三轮都未比对上的 reads"
"SEQKIT"grep−j{SEQKIT}" grep -j {ST_THREADS} -f "R3UNMAPPEDIDSCLEAN""{R3_UNMAPPED_IDS_CLEAN}" "{R2_UNMAPPED_FASTQ}" -o "${R3_UNMAPPED_FASTQ}"

echo "[R3] 最终未比对 FASTQ：R3UNMAPPEDFASTQ""{R3_UNMAPPED_FASTQ}" "{SEQKIT}" stats "${R3_UNMAPPED_FASTQ}"

R3 只删除原始 SAM 和未排序 BAM

echo "[R3] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "R3SAM""{R3_SAM}" "{R3_BAM}"

echo "=== 全流程完成（map-ont → asm20 → planA-mapont；三轮均未比对的 reads 已输出）==="
echo "保留文件（每一轮）："
echo " * R1SORTEDBAM及其.bai"echo"∗{R1_SORTED_BAM} 及其 .bai" echo " * {R1_UNMAPPED_BAM}"
echo " * R2SORTEDBAM及其.bai"echo"∗{R2_SORTED_BAM} 及其 .bai" echo " * {R2_UNMAPPED_BAM}"
echo " * R3SORTEDBAM及其.bai"echo"∗{R3_SORTED_BAM} 及其 .bai" echo " * {R3_UNMAPPED_BAM}" 我们现在需要修改这个代码，调用的minimap2为/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2， 其余的软件位置不变 /DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2/SV-test-fastq/Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq \ >/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2/Col-coty-sup-Chr1-SV-cs.sam 处理的文件名字以及风格如上，同时我们也处理Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq 以及Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq 结果名字命名风格类似，同时也经历三轮比对，但比对时候只保留--cs=long 去掉--MD 因为这两个tag冲突，请你根据这要求进行修改吧

思考

Clarifying file modifications for script update

The user showed paths for their new FASTQ files, like Chr1, Chr2, and Chr3, and asked for changes to the script to process those files. They also want the resulting SAM file names formatted a specific way. However, they only referenced a single final SAM output for Chr1, so I wonder if that's the final output after all three rounds of mapping or just for Round 1.

Understanding modifications needed for script update

It seems like the user wants the pipeline modified to process the new chromosome-specific FASTQ files, using three rounds of alignment while keeping only the --cs=long tag and removing --MD due to a conflict. The biggest question is whether I'll structure the pipeline to loop over the three chromosome FASTQs or just modify their paths.

Clarifying pipeline and file naming conventions

It looks like the user wants to align FASTQ files for each chromosome through a 3-round pipeline, and the output files need specific naming — for instance, using Col-coty-sup-Chr1-SV-cs.sam for the final SAM. It seems I'll need to adjust the script to account for this new naming structure, parsing the chromosome info from the file path and aligning it to their preferred format. The OUTDIR seems unchanged.

Adjusting pipeline to align FASTQs and output naming

It seems like the user wants the output SAM file named Col-coty-sup-Chr1-SV-cs.sam, but the script typically outputs sorted BAM files. They also want to process all FASTQ files in the SV-test-fastq subdirectory. I’ll adapt the script to keep the original output types (like sorted .bam) and create a final optional SAM with the desired tag.

Updating pipeline with new naming and mapping rules

The user wants to ensure the pipeline uses only the cs tag in minimap2 (no MD tag) to avoid conflicts. They also want to adjust sample naming to "Col-coty-sup" instead of "Col-coty-dorado" and map each chromosome's SV FASTQ individually. I’ll adapt the script to loop over the different chromosome files, process them, and adjust the sample naming as requested.

Modifying script for per-chromosome processing

The user wants the script to handle per-chromosome mapping, keeping output paths and sample names in a specific format, with the only modification being the removal of the --MD tag. I'll modify the script to loop over the chromosomes and adjust input/output paths and sample names, applying the requested changes for each chromosome's FASTQ input.

Designing the script with the correct naming and rounds

I need to create a script that runs multiple rounds of mapping, adjusting input paths and output names as per the user's instructions. I'll set the MINIMAP2 path, adjust the naming convention to match requested "Col-coty-sup-Chr1-SV-cs.sam" format, and ensure the script properly loops through all chromosomes. I'll apply changes to remove --MD, finalize the sample names, and generate the rounds using appropriate output names.

Finalizing script with clear input/output for per-chromosomes

I’ll keep the script simple, focusing on modifying variables for each chromosome input. I'll set a placeholder for IN_FASTQ and reflect the sorting you requested. For Chr2 and Chr3, the user can adjust IN_FASTQ and SAMPLE. Keeping the script flexible but working with generalized instructions is key.

Thought for 1m 3s

回答

我直接按你的要求把脚本改好：

  • minimap2 改成：/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2
  • 所有比对里 只保留 --cs=long，去掉 --MD
  • 输入例子用你给的 Chr1 FASTQ：
    /DataZhai/.../SV-test-fastq/Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
  • 输出目录仍然是原来的 minimap2-Col-CC-V2 目录
  • SAMPLE 改成 Col-coty-sup-Chr1-SV，输出命名风格类似你举的例子，仍保留三轮比对

对 Chr2、Chr3 只需要改两行：IN_FASTQ 和 SAMPLE，下面我也写了示例。

修改后的完整脚本（Chr1 示例）

bash
#!/usr/bin/env bash
set -euo pipefail

###############################################################################
# 线程数
###############################################################################
MM2_THREADS=36
ST_THREADS=36

###############################################################################
# 软件绝对路径
###############################################################################
MINIMAP2="/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2"
SAMTOOLS="/public/Zhaijx/wangz/Condaenvir/envs/samtools/bin/samtools"
SEQKIT="/public/Zhaijx/wangz/Condaenvir/envs/seqkit/bin/seqkit"

###############################################################################
# 参考基因组（Col-CC-V2）
###############################################################################
REF="/DataZhai/wangzh/genome/Col-CC-V2/Col-CC-V2.fasta"

###############################################################################
# 原始输入 FASTQ（此处示例为 Chr1 的 SV FASTQ）
#   Chr1: Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
#   Chr2: Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq
#   Chr3: Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq
###############################################################################
IN_FASTQ="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2/SV-test-fastq/Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq"

###############################################################################
# 输出目录
###############################################################################
OUTDIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2"
mkdir -p "${OUTDIR}"

###############################################################################
# sample 名，用来拼接输出文件前缀
#   这里示例：Col-coty-sup-Chr1-SV
#   如果处理 Chr2/Chr3，建议改为：
#      Chr2: Col-coty-sup-Chr2-SV
#      Chr3: Col-coty-sup-Chr3-SV
###############################################################################
SAMPLE="Col-coty-sup-Chr1-SV"
BASE="${OUTDIR}/${SAMPLE}-Col-CC-V2"

###############################################################################
# Round 1：minimap2 map-ont 对原始 FASTQ
###############################################################################
R1_PREFIX="${BASE}-r1-mapont"
R1_SAM="${R1_PREFIX}.sam"
R1_BAM="${R1_PREFIX}.bam"
R1_SORTED_BAM="${R1_PREFIX}-sorted.bam"
R1_UNMAPPED_BAM="${R1_PREFIX}-sorted-unmapped.bam"
R1_UNMAPPED_IDS="${R1_PREFIX}-sorted.unmapped_read_ids.txt"
R1_UNMAPPED_IDS_CLEAN="${R1_PREFIX}-sorted.unmapped_read_ids.clean.txt"
R1_UNMAPPED_FASTQ="${R1_PREFIX}.unmapped.fq.gz"

echo "[R1] minimap2 map-ont: ${IN_FASTQ}"
"${MINIMAP2}" -x map-ont -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${IN_FASTQ}" > "${R1_SAM}"

echo "[R1] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R1_BAM}" "${R1_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R1_SORTED_BAM}" "${R1_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R1_SORTED_BAM}"

echo "[R1] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R1_SORTED_BAM}" -o "${R1_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R1_UNMAPPED_BAM}" | awk '{print $1}' > "${R1_UNMAPPED_IDS}"

tr -d '\r' < "${R1_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R1_UNMAPPED_IDS_CLEAN}"

echo "[R1] 未比对 ID 行数：$(wc -l < "${R1_UNMAPPED_IDS_CLEAN}")"

echo "[R1] 使用 seqkit 从原始 FASTQ 抽取未比对 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R1_UNMAPPED_IDS_CLEAN}" "${IN_FASTQ}" -o "${R1_UNMAPPED_FASTQ}"

echo "[R1] R1_unmapped FASTQ 统计："
"${SEQKIT}" stats "${R1_UNMAPPED_FASTQ}"

# R1 只删除原始 SAM 和未排序 BAM
echo "[R1] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R1_SAM}" "${R1_BAM}"

###############################################################################
# Round 2：minimap2 asm20，对 R1 未比对 reads
###############################################################################
R2_PREFIX="${BASE}-r2-asm20"
R2_SAM="${R2_PREFIX}.sam"
R2_BAM="${R2_PREFIX}.bam"
R2_SORTED_BAM="${R2_PREFIX}-sorted.bam"
R2_UNMAPPED_BAM="${R2_PREFIX}-sorted-unmapped.bam"
R2_UNMAPPED_IDS="${R2_PREFIX}-sorted.unmapped_read_ids.txt"
R2_UNMAPPED_IDS_CLEAN="${R2_PREFIX}-sorted.unmapped_read_ids.clean.txt"
R2_UNMAPPED_FASTQ="${R2_PREFIX}.unmapped.fq.gz"

echo "[R2] minimap2 asm20: ${R1_UNMAPPED_FASTQ}"
"${MINIMAP2}" -x asm20 -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${R1_UNMAPPED_FASTQ}" > "${R2_SAM}"

echo "[R2] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R2_BAM}" "${R2_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R2_SORTED_BAM}" "${R2_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R2_SORTED_BAM}"

echo "[R2] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R2_SORTED_BAM}" -o "${R2_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R2_UNMAPPED_BAM}" | awk '{print $1}' > "${R2_UNMAPPED_IDS}"

tr -d '\r' < "${R2_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R2_UNMAPPED_IDS_CLEAN}"

echo "[R2] 未比对 ID 行数：$(wc -l < "${R2_UNMAPPED_IDS_CLEAN}")"

echo "[R2] 使用 seqkit 从 R1_unmapped FASTQ 抽取 R2 未比对 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R2_UNMAPPED_IDS_CLEAN}" "${R1_UNMAPPED_FASTQ}" -o "${R2_UNMAPPED_FASTQ}"

echo "[R2] R2_unmapped FASTQ 统计："
"${SEQKIT}" stats "${R2_UNMAPPED_FASTQ}"

# R2 只删除原始 SAM 和未排序 BAM
echo "[R2] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R2_SAM}" "${R2_BAM}"

###############################################################################
# Round 3：plan A（自定义 map-ont），对 R2 未比对 reads
###############################################################################
R3_PREFIX="${BASE}-r3-planA-mapont"
R3_SAM="${R3_PREFIX}.sam"
R3_BAM="${R3_PREFIX}.bam"
R3_SORTED_BAM="${R3_PREFIX}-sorted.bam"
R3_UNMAPPED_BAM="${R3_PREFIX}-sorted-unmapped.bam"
R3_UNMAPPED_IDS="${R3_PREFIX}-sorted.unmapped_read_ids.txt"
R3_UNMAPPED_IDS_CLEAN="${R3_PREFIX}-sorted.unmapped_read_ids.clean.txt"
# 最终三轮都未比对上的 reads
R3_UNMAPPED_FASTQ="${R3_PREFIX}.unmapped.fq.gz"

echo "[R3][planA] minimap2 map-ont with tuned params: ${R2_UNMAPPED_FASTQ}"
"${MINIMAP2}" -x map-ont -a --cs=long \
  -k13 -w7 \
  -r2k,60k -g8k \
  -U40,700 \
  -s70 -z600,200 \
  -N30 -t ${MM2_THREADS} \
  "${REF}" "${R2_UNMAPPED_FASTQ}" > "${R3_SAM}"

echo "[R3] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R3_BAM}" "${R3_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R3_SORTED_BAM}" "${R3_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R3_SORTED_BAM}"

echo "[R3] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R3_SORTED_BAM}" -o "${R3_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R3_UNMAPPED_BAM}" | awk '{print $1}' > "${R3_UNMAPPED_IDS}"

tr -d '\r' < "${R3_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R3_UNMAPPED_IDS_CLEAN}"

echo "[R3] 未比对 ID 行数：$(wc -l < "${R3_UNMAPPED_IDS_CLEAN}")"

echo "[R3] 使用 seqkit 从 R2_unmapped FASTQ 抽取三轮都未比对上的 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R3_UNMAPPED_IDS_CLEAN}" "${R2_UNMAPPED_FASTQ}" -o "${R3_UNMAPPED_FASTQ}"

echo "[R3] 最终未比对 FASTQ：${R3_UNMAPPED_FASTQ}"
"${SEQKIT}" stats "${R3_UNMAPPED_FASTQ}"

# R3 只删除原始 SAM 和未排序 BAM
echo "[R3] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R3_SAM}" "${R3_BAM}"

echo "=== 全流程完成（map-ont → asm20 → planA-mapont；三轮均未比对的 reads 已输出）==="
echo "保留文件（每一轮）："
echo "  * ${R1_SORTED_BAM} 及其 .bai"
echo "  * ${R1_UNMAPPED_BAM}"
echo "  * ${R2_SORTED_BAM} 及其 .bai"
echo "  * ${R2_UNMAPPED_BAM}"
echo "  * ${R3_SORTED_BAM} 及其 .bai"
echo "  * ${R3_UNMAPPED_BAM}"

如何处理 Chr2 / Chr3

对于 Chr2、Chr3，你可以复制这份脚本两份，分别改两行即可：

Chr2 示例

bash
IN_FASTQ="/DataZhai/wangzh/2024_11_ATXR5/.../SV-test-fastq/Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq"
SAMPLE="Col-coty-sup-Chr2-SV"

Chr3 示例

bash
IN_FASTQ="/DataZhai/wangzh/2024_11_ATXR5/.../SV-test-fastq/Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq"
SAMPLE="Col-coty-sup-Chr3-SV"

这样三份脚本分别跑 Chr1 / Chr2 / Chr3，都用新的 minimap2 路径，并且 只写 --cs=long 不再写 --MD，三轮比对逻辑保持不变。

如果你希望最后某一轮（比如 R1 或 R3）的 SAM 名字完全变成类似
Col-coty-sup-Chr1-SV-cs.sam，我也可以帮你再把前缀变量改成这个精确名字。

问题

我希望你可以写一个循环，一次性依次处理三个文件，三个文件名字只有Chr的数字不同

思考

Considering user’s request for bash script modifications

The user wants me to modify the bash script for their minimap2 pipeline. They want to use a loop to process files with names that differ only by chromosome number. They also want consistent naming. I'll need to loop over chromosomes and make sure each output file embeds the correct ChrN, matching the naming style they’ve provided. I'll also ensure I’m keeping the details of the three rounds of mapping in mind.

Adjusting pipeline to include loops and consistent naming

The user has accepted the pipeline’s structure but wants loops now. They want the naming style similar to previous examples, but now with chromosome numbers. I’ll create a loop like for chr in 1 2 3, using Col-coty-sup-Chr${CHR}-SV as the sample name. I'll make sure to keep the setup DRY, ensuring variable names update inside the loop and maintain safety settings like set -euo pipefail.

Creating the loop-based script for minimap2

I’ll produce a script using a loop to process chromosomes 1, 2, and 3. The sample name will update dynamically for each chromosome. I’ll include checks for correct paths and ensure the --MD tag is removed from all minimap2 calls. I’ll keep it simple, respond in Chinese, and encapsulate the script in a code block for easy copy-pasting.

Finalizing script with naming and multiple rounds

The user wants similar naming, but it seems they're using a 3-round pipeline producing multiple SAM files per round. They didn't request the final file to specifically include -cs.sam, but I think aligning the naming to their style is important. I'll modify the BASE to include -cs, which should give the desired format without overcomplicating things. I’ll keep everything else consistent with the pipeline structure.

Clarifying the user's request and finalizing naming

The user wants a loop to handle the three files with different chromosome numbers. They didn't explicitly ask for a specific cs.sam naming convention, so I should proceed with the earlier naming strategy. I'll reuse the pipeline code inside a loop, updating the IN_FASTQ and sample names accordingly.

Thought for 36s

回答

可以的，我们把这三条 Chr 的 FASTQ 放到一个循环里，一次性按相同流程跑完三轮比对。

下面这个脚本会依次处理：

  • Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
  • Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq
  • Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq

同时满足你之前所有要求：

  • 使用指定的 minimap2：/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2
  • samtools / seqkit 路径不变
  • 三轮比对 (map-ont → asm20 → 调参 map-ont) 全部保留
  • 所有 minimap2 命令 只有 --cs=long，去掉 --MD
  • 命名风格：SAMPLE="Col-coty-sup-Chr${CHR}-SV"，输出前缀类似之前：Col-coty-sup-Chr1-SV-Col-CC-V2-r1-mapont.* 等

循环版完整脚本

bash
#!/usr/bin/env bash
set -euo pipefail

###############################################################################
# 线程数
###############################################################################
MM2_THREADS=36
ST_THREADS=36

###############################################################################
# 软件绝对路径
###############################################################################
MINIMAP2="/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2"
SAMTOOLS="/public/Zhaijx/wangz/Condaenvir/envs/samtools/bin/samtools"
SEQKIT="/public/Zhaijx/wangz/Condaenvir/envs/seqkit/bin/seqkit"

###############################################################################
# 参考基因组（Col-CC-V2）
###############################################################################
REF="/DataZhai/wangzh/genome/Col-CC-V2/Col-CC-V2.fasta"

###############################################################################
# 输入/输出目录
###############################################################################
SV_DIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2/SV-test-fastq"
OUTDIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2"
mkdir -p "${OUTDIR}"

###############################################################################
# 依次处理 Chr1 / Chr2 / Chr3
###############################################################################
for CHR in 1 2 3; do
  echo "=============================="
  echo " 开始处理 Chr${CHR}"
  echo "=============================="

  ###########################################################################
  # 当前染色体对应的输入 FASTQ & SAMPLE 名
  #   输入：Col-coty-dorado-minimap2-Col-CC-V2-Chr${CHR}-SV.fastq
  #   输出前缀 SAMPLE：Col-coty-sup-Chr${CHR}-SV
  ###########################################################################
  IN_FASTQ="${SV_DIR}/Col-coty-dorado-minimap2-Col-CC-V2-Chr${CHR}-SV.fastq"
  SAMPLE="Col-coty-sup-Chr${CHR}-SV"
  BASE="${OUTDIR}/${SAMPLE}-Col-CC-V2"

  if [[ ! -s "${IN_FASTQ}" ]]; then
    echo "ERROR: 输入 FASTQ 不存在或为空: ${IN_FASTQ}" >&2
    exit 1
  fi

  echo "[Chr${CHR}] 输入 FASTQ: ${IN_FASTQ}"
  echo "[Chr${CHR}] SAMPLE    : ${SAMPLE}"
  echo "[Chr${CHR}] BASE 前缀 : ${BASE}"
  echo

  #############################################################################
  # Round 1：minimap2 map-ont 对原始 FASTQ
  #############################################################################
  R1_PREFIX="${BASE}-r1-mapont"
  R1_SAM="${R1_PREFIX}.sam"
  R1_BAM="${R1_PREFIX}.bam"
  R1_SORTED_BAM="${R1_PREFIX}-sorted.bam"
  R1_UNMAPPED_BAM="${R1_PREFIX}-sorted-unmapped.bam"
  R1_UNMAPPED_IDS="${R1_PREFIX}-sorted.unmapped_read_ids.txt"
  R1_UNMAPPED_IDS_CLEAN="${R1_PREFIX}-sorted.unmapped_read_ids.clean.txt"
  R1_UNMAPPED_FASTQ="${R1_PREFIX}.unmapped.fq.gz"

  echo "[Chr${CHR}][R1] minimap2 map-ont: ${IN_FASTQ}"
  "${MINIMAP2}" -x map-ont -a --cs=long \
    -t ${MM2_THREADS} \
    "${REF}" "${IN_FASTQ}" > "${R1_SAM}"

  echo "[Chr${CHR}][R1] SAM -> BAM & sort"
  "${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R1_BAM}" "${R1_SAM}"
  "${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R1_SORTED_BAM}" "${R1_BAM}"
  "${SAMTOOLS}" index -@ ${ST_THREADS} "${R1_SORTED_BAM}"

  echo "[Chr${CHR}][R1] 提取未比对 reads 的 ID (flag 4)"
  "${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R1_SORTED_BAM}" -o "${R1_UNMAPPED_BAM}"
  "${SAMTOOLS}" view -@ ${ST_THREADS} "${R1_UNMAPPED_BAM}" | awk '{print $1}' > "${R1_UNMAPPED_IDS}"

  tr -d '\r' < "${R1_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R1_UNMAPPED_IDS_CLEAN}"

  echo "[Chr${CHR}][R1] 未比对 ID 行数：$(wc -l < "${R1_UNMAPPED_IDS_CLEAN}")"

  echo "[Chr${CHR}][R1] 使用 seqkit 从原始 FASTQ 抽取未比对 reads"
  "${SEQKIT}" grep -j ${ST_THREADS} -f "${R1_UNMAPPED_IDS_CLEAN}" "${IN_FASTQ}" -o "${R1_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R1] R1_unmapped FASTQ 统计："
  "${SEQKIT}" stats "${R1_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R1] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
  rm -f "${R1_SAM}" "${R1_BAM}"
  echo

  #############################################################################
  # Round 2：minimap2 asm20，对 R1 未比对 reads
  #############################################################################
  R2_PREFIX="${BASE}-r2-asm20"
  R2_SAM="${R2_PREFIX}.sam"
  R2_BAM="${R2_PREFIX}.bam"
  R2_SORTED_BAM="${R2_PREFIX}-sorted.bam"
  R2_UNMAPPED_BAM="${R2_PREFIX}-sorted-unmapped.bam"
  R2_UNMAPPED_IDS="${R2_PREFIX}-sorted.unmapped_read_ids.txt"
  R2_UNMAPPED_IDS_CLEAN="${R2_PREFIX}-sorted.unmapped_read_ids.clean.txt"
  R2_UNMAPPED_FASTQ="${R2_PREFIX}.unmapped.fq.gz"

  echo "[Chr${CHR}][R2] minimap2 asm20: ${R1_UNMAPPED_FASTQ}"
  "${MINIMAP2}" -x asm20 -a --cs=long \
    -t ${MM2_THREADS} \
    "${REF}" "${R1_UNMAPPED_FASTQ}" > "${R2_SAM}"

  echo "[Chr${CHR}][R2] SAM -> BAM & sort"
  "${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R2_BAM}" "${R2_SAM}"
  "${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R2_SORTED_BAM}" "${R2_BAM}"
  "${SAMTOOLS}" index -@ ${ST_THREADS} "${R2_SORTED_BAM}"

  echo "[Chr${CHR}][R2] 提取未比对 reads 的 ID (flag 4)"
  "${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R2_SORTED_BAM}" -o "${R2_UNMAPPED_BAM}"
  "${SAMTOOLS}" view -@ ${ST_THREADS} "${R2_UNMAPPED_BAM}" | awk '{print $1}' > "${R2_UNMAPPED_IDS}"

  tr -d '\r' < "${R2_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R2_UNMAPPED_IDS_CLEAN}"

  echo "[Chr${CHR}][R2] 未比对 ID 行数：$(wc -l < "${R2_UNMAPPED_IDS_CLEAN}")"

  echo "[Chr${CHR}][R2] 使用 seqkit 从 R1_unmapped FASTQ 抽取 R2 未比对 reads"
  "${SEQKIT}" grep -j ${ST_THREADS} -f "${R2_UNMAPPED_IDS_CLEAN}" "${R1_UNMAPPED_FASTQ}" -o "${R2_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R2] R2_unmapped FASTQ 统计："
  "${SEQKIT}" stats "${R2_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R2] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
  rm -f "${R2_SAM}" "${R2_BAM}"
  echo

  #############################################################################
  # Round 3：plan A（自定义 map-ont），对 R2 未比对 reads
  #############################################################################
  R3_PREFIX="${BASE}-r3-planA-mapont"
  R3_SAM="${R3_PREFIX}.sam"
  R3_BAM="${R3_PREFIX}.bam"
  R3_SORTED_BAM="${R3_PREFIX}-sorted.bam"
  R3_UNMAPPED_BAM="${R3_PREFIX}-sorted-unmapped.bam"
  R3_UNMAPPED_IDS="${R3_PREFIX}-sorted.unmapped_read_ids.txt"
  R3_UNMAPPED_IDS_CLEAN="${R3_PREFIX}-sorted.unmapped_read_ids.clean.txt"
  # 最终三轮都未比对上的 reads
  R3_UNMAPPED_FASTQ="${R3_PREFIX}.unmapped.fq.gz"

  echo "[Chr${CHR}][R3][planA] minimap2 map-ont with tuned params: ${R2_UNMAPPED_FASTQ}"
  "${MINIMAP2}" -x map-ont -a --cs=long \
    -k13 -w7 \
    -r2k,60k -g8k \
    -U40,700 \
    -s70 -z600,200 \
    -N30 -t ${MM2_THREADS} \
    "${REF}" "${R2_UNMAPPED_FASTQ}" > "${R3_SAM}"

  echo "[Chr${CHR}][R3] SAM -> BAM & sort"
  "${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R3_BAM}" "${R3_SAM}"
  "${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R3_SORTED_BAM}" "${R3_BAM}"
  "${SAMTOOLS}" index -@ ${ST_THREADS} "${R3_SORTED_BAM}"

  echo "[Chr${CHR}][R3] 提取未比对 reads 的 ID (flag 4)"
  "${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R3_SORTED_BAM}" -o "${R3_UNMAPPED_BAM}"
  "${SAMTOOLS}" view -@ ${ST_THREADS} "${R3_UNMAPPED_BAM}" | awk '{print $1}' > "${R3_UNMAPPED_IDS}"

  tr -d '\r' < "${R3_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R3_UNMAPPED_IDS_CLEAN}"

  echo "[Chr${CHR}][R3] 未比对 ID 行数：$(wc -l < "${R3_UNMAPPED_IDS_CLEAN}")"

  echo "[Chr${CHR}][R3] 使用 seqkit 从 R2_unmapped FASTQ 抽取三轮都未比对上的 reads"
  "${SEQKIT}" grep -j ${ST_THREADS} -f "${R3_UNMAPPED_IDS_CLEAN}" "${R2_UNMAPPED_FASTQ}" -o "${R3_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R3] 最终未比对 FASTQ：${R3_UNMAPPED_FASTQ}"
  "${SEQKIT}" stats "${R3_UNMAPPED_FASTQ}"

  echo "[Chr${CHR}][R3] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
  rm -f "${R3_SAM}" "${R3_BAM}"

  echo "=== Chr${CHR} 三轮比对完成 ==="
  echo "保留文件（每一轮）："
  echo "  * ${R1_SORTED_BAM} 及其 .bai"
  echo "  * ${R1_UNMAPPED_BAM}"
  echo "  * ${R2_SORTED_BAM} 及其 .bai"
  echo "  * ${R2_UNMAPPED_BAM}"
  echo "  * ${R3_SORTED_BAM} 及其 .bai"
  echo "  * ${R3_UNMAPPED_BAM}"
  echo
done

echo "=== 全部 Chr1/2/3 处理完成 ==="

如果你想让某一轮（比如 R1）额外输出一个简短名字的 SAM（比如 Col-coty-sup-Chr1-SV-cs.sam）做主结果，我也可以在循环里帮你加一个 cp / ln -s 生成这个别名。

问题

#!/usr/bin/env bash
set -euo pipefail

###############################################################################

线程数

###############################################################################
MM2_THREADS=36
ST_THREADS=36

###############################################################################

软件绝对路径

###############################################################################
MINIMAP2="/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2"
SAMTOOLS="/public/Zhaijx/wangz/Condaenvir/envs/samtools/bin/samtools"
SEQKIT="/public/Zhaijx/wangz/Condaenvir/envs/seqkit/bin/seqkit"

###############################################################################

参考基因组（Col-CC-V2）

###############################################################################
REF="/DataZhai/wangzh/genome/Col-CC-V2/Col-CC-V2.fasta"

###############################################################################

输出目录

###############################################################################
OUTDIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2"
mkdir -p "${OUTDIR}"

###############################################################################

定义处理函数

###############################################################################
process_fastq() {
local IN_FASTQ="1"localCHROMOSOME="1" local CHROMOSOME="2"

text
# 从输入文件名提取基本信息
local FILENAME=$(basename "${IN_FASTQ}" .fastq)
local SAMPLE="Col-coty-sup-${CHROMOSOME}-SV"
local BASE="${OUTDIR}/${SAMPLE}-Col-CC-V2"

echo "========================================"
echo "开始处理: ${IN_FASTQ}"
echo "染色体: ${CHROMOSOME}"
echo "样本名: ${SAMPLE}"
echo "========================================"

###############################################################################
# Round 1：minimap2 map-ont 对原始 FASTQ
###############################################################################
R1_PREFIX="${BASE}-r1-mapont"
R1_SAM="${R1_PREFIX}.sam"
R1_BAM="${R1_PREFIX}.bam"
R1_SORTED_BAM="${R1_PREFIX}-sorted.bam"
R1_UNMAPPED_BAM="${R1_PREFIX}-sorted-unmapped.bam"
R1_UNMAPPED_IDS="${R1_PREFIX}-sorted.unmapped_read_ids.txt"
R1_UNMAPPED_IDS_CLEAN="${R1_PREFIX}-sorted.unmapped_read_ids.clean.txt"
R1_UNMAPPED_FASTQ="${R1_PREFIX}.unmapped.fq.gz"

echo "[R1] minimap2 map-ont: ${IN_FASTQ}"
"${MINIMAP2}" -x map-ont -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${IN_FASTQ}" > "${R1_SAM}"

echo "[R1] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R1_BAM}" "${R1_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R1_SORTED_BAM}" "${R1_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R1_SORTED_BAM}"

echo "[R1] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R1_SORTED_BAM}" -o "${R1_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R1_UNMAPPED_BAM}" | awk '{print $1}' > "${R1_UNMAPPED_IDS}"

tr -d '\r' < "${R1_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R1_UNMAPPED_IDS_CLEAN}"

echo "[R1] 未比对 ID 行数：$(wc -l < "${R1_UNMAPPED_IDS_CLEAN}")"

echo "[R1] 使用 seqkit 从原始 FASTQ 抽取未比对 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R1_UNMAPPED_IDS_CLEAN}" "${IN_FASTQ}" -o "${R1_UNMAPPED_FASTQ}"

echo "[R1] R1_unmapped FASTQ 统计："
"${SEQKIT}" stats "${R1_UNMAPPED_FASTQ}"

# R1 只删除原始 SAM 和未排序 BAM
echo "[R1] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R1_SAM}" "${R1_BAM}"

###############################################################################
# Round 2：minimap2 asm20，对 R1 未比对 reads
###############################################################################
R2_PREFIX="${BASE}-r2-asm20"
R2_SAM="${R2_PREFIX}.sam"
R2_BAM="${R2_PREFIX}.bam"
R2_SORTED_BAM="${R2_PREFIX}-sorted.bam"
R2_UNMAPPED_BAM="${R2_PREFIX}-sorted-unmapped.bam"
R2_UNMAPPED_IDS="${R2_PREFIX}-sorted.unmapped_read_ids.txt"
R2_UNMAPPED_IDS_CLEAN="${R2_PREFIX}-sorted.unmapped_read_ids.clean.txt"
R2_UNMAPPED_FASTQ="${R2_PREFIX}.unmapped.fq.gz"

echo "[R2] minimap2 asm20: ${R1_UNMAPPED_FASTQ}"
"${MINIMAP2}" -x asm20 -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${R1_UNMAPPED_FASTQ}" > "${R2_SAM}"

echo "[R2] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R2_BAM}" "${R2_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R2_SORTED_BAM}" "${R2_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R2_SORTED_BAM}"

echo "[R2] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R2_SORTED_BAM}" -o "${R2_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R2_UNMAPPED_BAM}" | awk '{print $1}' > "${R2_UNMAPPED_IDS}"

tr -d '\r' < "${R2_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R2_UNMAPPED_IDS_CLEAN}"

echo "[R2] 未比对 ID 行数：$(wc -l < "${R2_UNMAPPED_IDS_CLEAN}")"

echo "[R2] 使用 seqkit 从 R1_unmapped FASTQ 抽取 R2 未比对 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R2_UNMAPPED_IDS_CLEAN}" "${R1_UNMAPPED_FASTQ}" -o "${R2_UNMAPPED_FASTQ}"

echo "[R2] R2_unmapped FASTQ 统计："
"${SEQKIT}" stats "${R2_UNMAPPED_FASTQ}"

# R2 只删除原始 SAM 和未排序 BAM
echo "[R2] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R2_SAM}" "${R2_BAM}"

###############################################################################
# Round 3：plan A（自定义 map-ont），对 R2 未比对 reads
###############################################################################
R3_PREFIX="${BASE}-r3-planA-mapont"
R3_SAM="${R3_PREFIX}.sam"
R3_BAM="${R3_PREFIX}.bam"
R3_SORTED_BAM="${R3_PREFIX}-sorted.bam"
R3_UNMAPPED_BAM="${R3_PREFIX}-sorted-unmapped.bam"
R3_UNMAPPED_IDS="${R3_PREFIX}-sorted.unmapped_read_ids.txt"
R3_UNMAPPED_IDS_CLEAN="${R3_PREFIX}-sorted.unmapped_read_ids.clean.txt"
# 最终三轮都未比对上的 reads
R3_UNMAPPED_FASTQ="${R3_PREFIX}.unmapped.fq.gz"

echo "[R3][planA] minimap2 map-ont with tuned params: ${R2_UNMAPPED_FASTQ}"
"${MINIMAP2}" -x map-ont -a --cs=long \
  -k13 -w7 \
  -r2k,60k -g8k \
  -U40,700 \
  -s70 -z600,200 \
  -N30 -t ${MM2_THREADS} \
  "${REF}" "${R2_UNMAPPED_FASTQ}" > "${R3_SAM}"

echo "[R3] SAM -> BAM & sort"
"${SAMTOOLS}" view  -@ ${ST_THREADS} -b -o "${R3_BAM}" "${R3_SAM}"
"${SAMTOOLS}" sort  -@ ${ST_THREADS} -o "${R3_SORTED_BAM}" "${R3_BAM}"
"${SAMTOOLS}" index -@ ${ST_THREADS} "${R3_SORTED_BAM}"

echo "[R3] 提取未比对 reads 的 ID (flag 4)"
"${SAMTOOLS}" view -@ ${ST_THREADS} -b -f 4 "${R3_SORTED_BAM}" -o "${R3_UNMAPPED_BAM}"
"${SAMTOOLS}" view -@ ${ST_THREADS} "${R3_UNMAPPED_BAM}" | awk '{print $1}' > "${R3_UNMAPPED_IDS}"

tr -d '\r' < "${R3_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R3_UNMAPPED_IDS_CLEAN}"

echo "[R3] 未比对 ID 行数：$(wc -l < "${R3_UNMAPPED_IDS_CLEAN}")"

echo "[R3] 使用 seqkit 从 R2_unmapped FASTQ 抽取三轮都未比对上的 reads"
"${SEQKIT}" grep -j ${ST_THREADS} -f "${R3_UNMAPPED_IDS_CLEAN}" "${R2_UNMAPPED_FASTQ}" -o "${R3_UNMAPPED_FASTQ}"

echo "[R3] 最终未比对 FASTQ：${R3_UNMAPPED_FASTQ}"
"${SEQKIT}" stats "${R3_UNMAPPED_FASTQ}"

# R3 只删除原始 SAM 和未排序 BAM
echo "[R3] 删除原始 SAM/BAM，保留 sorted.bam / bai / unmapped.bam"
rm -f "${R3_SAM}" "${R3_BAM}"

echo "=== ${CHROMOSOME} 处理完成 ==="
echo "保留文件："
echo "  * ${R1_SORTED_BAM} 及其 .bai"
echo "  * ${R1_UNMAPPED_BAM}"
echo "  * ${R2_SORTED_BAM} 及其 .bai"
echo "  * ${R2_UNMAPPED_BAM}"
echo "  * ${R3_SORTED_BAM} 及其 .bai"
echo "  * ${R3_UNMAPPED_BAM}"
echo ""

}

###############################################################################

主程序：依次处理三个染色体

###############################################################################

定义三个染色体的FASTQ文件

FASTQ_DIR="/DataZhai/wangzh/2024_11_ATXR5/fastq/long/2024-11-dorado-ultra/2024-11-25-Zhai/Col-0/minimap2-Col-CC-V2/SV-test-fastq"

依次处理三个染色体

for CHR in Chr1 Chr2 Chr3; do
IN_FASTQ="FASTQDIR/Col−coty−dorado−minimap2−Col−CC−V2−{FASTQ_DIR}/Col-coty-dorado-minimap2-Col-CC-V2-{CHR}-SV.fastq"

text
# 检查文件是否存在
if [[ ! -f "${IN_FASTQ}" ]]; then
    echo "错误：文件不存在: ${IN_FASTQ}"
    continue
fi

# 调用处理函数
process_fastq "${IN_FASTQ}" "${CHR}"

done

echo "=== 所有染色体处理完成 ===" 请你帮忙仔细检查这段代码，并告诉我他的逻辑功能，以及能否正常运行

思考

Reviewing bash script logic

The script starts off well with settings like #!/usr/bin/env bash and set -euo pipefail, which ensures safe execution. It defines useful variables, such as paths and output directories. The function process_fastq() takes IN_FASTQ and CHROMOSOME inputs, though some variables like FILENAME are defined but not used, which is something to note. The SAMPLE variable seems correct as it's based on the chromosome, such as 'Col-coty-sup-Chr1-SV'.

Reviewing Round1 logic

In Round1, variables like R1_PREFIX are defined globally but not using the local keyword, making them accessible across function calls — though this means they could be overwritten in subsequent calls, which is okay as long as each call is sequential. The Minimap2 command is set correctly, and the samtools function looks solid for sorting and indexing. I also see that the stream is cleaned and sorted with awk and tr for accurate handling of unmapped IDs.

Reviewing rounds of processing for FASTQ files

In Round1, the script uses compressed .fq.gz files, with seqkit handling compression based on extension. Round2 follows a similar structure, processing the R1_UNMAPPED_FASTQ with asm20, while Round3 adds extra tuning like --cs=long for the mapping. The final step of the function echoes completion. The main logic loops over chromosomes, confirming the paths and naming, ensuring they align with previous inputs.

Checking subtle points and potential issues

The script is set up to continue execution even if ${IN_FASTQ} doesn't exist by printing an error. The function usage is also solid, with defined variables and no undefined errors. However, while the script doesn't explicitly check for the existence of commands like MINIMAP2 or SAMTOOLS, it would error if missing. Quoting CHROMOSOME appears okay, and since the script isn't handling multiple calls simultaneously, there's no concurrency issue. Lastly, I’ll briefly check for any off-by-one errors.

Reviewing nuances and potential concerns

In R3, using seqkit grep with R3_UNMAPPED_IDS_CLEAN is fine, but if there are no unmapped reads, the input files like ${R1_UNMAPPED_FASTQ} might still exist but be empty. Minimap2 can still run, but nothing will be mapped. If R1_UNMAPPED_IDS_CLEAN is empty, seqkit will output nothing, which is expected. Since Minimap2 supports .fq.gz files, there's no issue with compression. There are a few small tweaks, like making variables local to avoid side effects, but overall, the script structure seems solid.

Double-checking output naming and paths

The output and FASTQ directory paths are consistent, and the user’s example suggested a SAM file named Col-coty-sup-Chr1-SV-cs.sam. However, the script currently doesn’t create this exact output, instead generating a different SAM file (R1_SAM) that's deleted afterward. The user seems to just want a similar naming style, not necessarily the exact -cs.sam. The script loops over chromosomes correctly. Minor tweaks could ensure naming more closely matches the user’s request, but the logic seems sound.

Script logic check and potential improvements

The script seems good overall, though there are some minor points to note. The local FILENAME variable isn’t being used, though it might’ve been intended to extract the chromosome name from the file. A small style issue is the use of local only for some variables but not others. Also, if a FASTQ file is empty, seqkit stats might still run but won’t cause a failure. The shebang with /usr/bin/env works fine, and there's no issue with variable quoting. The script

Thought for 39s

回答

先说结论：
这段脚本逻辑是清晰的，能实现你说的“三个 Chr 文件依次跑三轮比对”的流程；只要路径、软件都存在，就可以正常运行。下面我帮你把它拆开讲一下具体做了什么，同时指出几个可以注意的小细节（不是致命错误）。

一、整体逻辑功能

  1. 全局设置

    • set -euo pipefail：遇到错误立即退出、未定义变量报错、管道中任一命令出错都视为失败（安全）。
    • 设定线程数：MM2_THREADS=36（minimap2），ST_THREADS=36（samtools / seqkit）。
    • 设定软件路径：
      • MINIMAP2="/DataZhai/wangzh/software/minimap2-2.30_x64-linux/minimap2"
      • SAMTOOLS="/public/Zhaijx/wangz/Condaenvir/envs/samtools/bin/samtools"
      • SEQKIT="/public/Zhaijx/wangz/Condaenvir/envs/seqkit/bin/seqkit"
    • 参考基因组：REF="/DataZhai/wangzh/genome/Col-CC-V2/Col-CC-V2.fasta"
    • 输出目录：OUTDIR="/.../minimap2-Col-CC-V2"，并 mkdir -p。

  2. 定义一个函数 process_fastq IN_FASTQ CHROMOSOME

    • 用来对单个 FASTQ（某一条染色体的 SV-FASTQ）执行三轮比对 + 提取未比对 reads 的流程。
    • 传入：
      • IN_FASTQ：完整路径，如 .../Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
      • CHROMOSOME：Chr1 / Chr2 / Chr3
    • 根据 CHR 构建 sample 名：
      SAMPLE="Col-coty-sup-${CHROMOSOME}-SV"
      比如 Chr1 → Col-coty-sup-Chr1-SV
    • 输出前缀：
      BASE="${OUTDIR}/${SAMPLE}-Col-CC-V2"
      例如：/…/Col-coty-sup-Chr1-SV-Col-CC-V2

  3. 主程序

    • 定义 FASTQ 所在目录：
      FASTQ_DIR="/.../SV-test-fastq"
    • 循环 for CHR in Chr1 Chr2 Chr3; do ... done
      • 构造对应的输入 FASTQ： IN_FASTQ="${FASTQ_DIR}/Col-coty-dorado-minimap2-Col-CC-V2-${CHR}-SV.fastq" → 即：
        • Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
        • Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq
        • Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq
      • 如果文件不存在：打印错误并 continue（跳过这个 Chr）。
      • 如果存在：process_fastq "${IN_FASTQ}" "${CHR}"
        → 顺序处理 Chr1 → Chr2 → Chr3。

二、单个 FASTQ 的三轮比对逻辑

在 process_fastq() 里，对每个输入 FASTQ 执行完全一样的流程，只是 CHROMOSOME/SAMPLE/BASE 不同。

1）Round 1：map-ont 对原始 FASTQ

  • 前缀：R1_PREFIX="${BASE}-r1-mapont"
  • 生成文件：
    • SAM：R1_SAM="${R1_PREFIX}.sam"
    • 排序后的 BAM：R1_SORTED_BAM="${R1_PREFIX}-sorted.bam"
    • 未比对 BAM：R1_UNMAPPED_BAM="${R1_PREFIX}-sorted-unmapped.bam"
    • 未比对 ID 列表（原始 & clean）：
      R1_UNMAPPED_IDS, R1_UNMAPPED_IDS_CLEAN
    • R1 未比对 FASTQ：R1_UNMAPPED_FASTQ="${R1_PREFIX}.unmapped.fq.gz"

流程：

  1. 比对：

    bash
    "${MINIMAP2}" -x map-ont -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${IN_FASTQ}" > "${R1_SAM}"
    • 用新的 minimap2 路径。
    • 只保留 --cs=long，没有 --MD，符合你说的“这两个 tag 冲突，去掉 --MD”。

  2. SAM → BAM → sort → index：

    bash
    samtools view  -@ ST_THREADS -b -o R1_BAM R1_SAM
samtools sort  -@ ST_THREADS -o R1_SORTED_BAM R1_BAM
samtools index -@ ST_THREADS R1_SORTED_BAM

  3. 提取未比对 reads（flag 4）：

    bash
    samtools view -@ ST_THREADS -b -f 4 R1_SORTED_BAM -o R1_UNMAPPED_BAM
samtools view -@ ST_THREADS R1_UNMAPPED_BAM | awk '{print $1}' > R1_UNMAPPED_IDS
tr -d '\r' < R1_UNMAPPED_IDS | awk 'length($0)>0' | LC_ALL=C sort -u > R1_UNMAPPED_IDS_CLEAN

  4. 用 seqkit 从原始 FASTQ 抽取未比对 reads：

    bash
    seqkit grep -j ST_THREADS -f R1_UNMAPPED_IDS_CLEAN IN_FASTQ -o R1_UNMAPPED_FASTQ

  5. 统计 + 清理：

    • seqkit stats R1_UNMAPPED_FASTQ
    • 删除中间 R1_SAM 和 R1_BAM，保留 sorted.bam / bai / unmapped.bam 和 unmapped.fq.gz。

2）Round 2：asm20 对 R1 未比对 FASTQ

  • 前缀：R2_PREFIX="${BASE}-r2-asm20"
  • 输入 FASTQ：R1_UNMAPPED_FASTQ
  • 其他逻辑和 R1 类似，只是：
    • 使用 minimap2 preset -x asm20
    • 输出文件名换成 R2 前缀。
    • 从 R1_unmapped.fq.gz 抽 R2 未比对 reads，写到 R2_UNMAPPED_FASTQ。

关键比对命令：

bash
"${MINIMAP2}" -x asm20 -a --cs=long \
  -t ${MM2_THREADS} \
  "${REF}" "${R1_UNMAPPED_FASTQ}" > "${R2_SAM}"

同样只有 --cs=long。

3）Round 3：自定义 map-ont（plan A）对 R2 未比对 FASTQ

  • 前缀：R3_PREFIX="${BASE}-r3-planA-mapont"

  • 输入 FASTQ：R2_UNMAPPED_FASTQ

  • 参数更激进：

    bash
    "${MINIMAP2}" -x map-ont -a --cs=long \
  -k13 -w7 \
  -r2k,60k -g8k \
  -U40,700 \
  -s70 -z600,200 \
  -N30 -t ${MM2_THREADS} \
  "${REF}" "${R2_UNMAPPED_FASTQ}" > "${R3_SAM}"

  • 后续流程同样：

    • SAM→BAM→sort→index
    • 提取 flag 4 → ID 去重 → seqkit grep → R3_UNMAPPED_FASTQ（三轮都没比上的 reads）
    • seqkit stats R3_UNMAPPED_FASTQ
    • 删除 R3_SAM & R3_BAM

最终保留的文件（每个 Chr）：

  • BASE-r1-mapont-sorted.bam 及 .bai
  • BASE-r1-mapont-sorted-unmapped.bam
  • BASE-r2-asm20-sorted.bam 及 .bai
  • BASE-r2-asm20-sorted-unmapped.bam
  • BASE-r3-planA-mapont-sorted.bam 及 .bai
  • BASE-r3-planA-mapont-sorted-unmapped.bam
  • 三轮最终未比对 FASTQ：BASE-r3-planA-mapont.unmapped.fq.gz

（三轮的 unmapped.fq.gz 也都会保留）

三、能否正常运行？可能的小问题/建议

1. 是否能正常跑？

在下面前提满足情况下，脚本可以正常运行：

  • 这三个输入 FASTQ 文件路径正确、文件存在：
    • .../Col-coty-dorado-minimap2-Col-CC-V2-Chr1-SV.fastq
    • .../Col-coty-dorado-minimap2-Col-CC-V2-Chr2-SV.fastq
    • .../Col-coty-dorado-minimap2-Col-CC-V2-Chr3-SV.fastq
  • MINIMAP2, SAMTOOLS, SEQKIT 路径都有效且具有执行权限。
  • REF 存在且格式正确。
  • 磁盘空间足够，因为会生成不少 BAM/FASTQ。

逻辑上没有明显 bug，变量在使用前都被赋值，set -u 不会踩雷。

2. 一些细节建议（非必须）

  1. 变量 local 化（风格问题，不影响正确性）
    在 process_fastq 里只给 IN_FASTQ 和 CHROMOSOME 加了 local，
    其它比如 R1_PREFIX / R2_PREFIX / R3_PREFIX 其实也可以 local，防止将来函数里面又调用其它函数时变量相互污染。现在这种写法在顺序单线程执行时也不会出错，只是代码更“干净”会更好。

  2. FILENAME 没有被使用

    bash
    local FILENAME=$(basename "${IN_FASTQ}" .fastq)

    这个变量后面没有用到，可以删掉，或者将来用它拼更复杂的名字。

  3. 0 个未比对 reads 的情况

    • 如果某一轮完全比对上，没有未比对 reads：
      • R*_UNMAPPED_IDS_CLEAN 会是空文件；
      • seqkit grep -f 空文件 会输出一个 0 read 的 FASTQ（或空文件）；
        minimap2 对空 FASTQ 一般能跑过去（只是不输出比对），所以整体不会挂。但如果你想“完全跳过后续轮次”，可以以后加判断，比如：
    bash
    if [[ ! -s "${R1_UNMAPPED_IDS_CLEAN}" ]]; then
    echo "R1 已经全部比对上，跳过 R2/R3"
    return 0
fi

    这属于优化，不是必须。

  4. 输出是否需要一个最终 SAM 名字（比如你最早说的 ...-cs.sam）
    当前脚本会把所有 *.sam 在对应轮次结束后删除，只留下 BAM 和 unmapped FASTQ。
    如果你确实还想要一个类似：

    text
    /.../Col-coty-sup-Chr1-SV-cs.sam

    作为“主结果”，可以在 R1 比对后加一行：

    bash
    cp "${R1_SAM}" "${OUTDIR}/${SAMPLE}-cs.sam"

    再删 R1_SAM。
    现在的脚本逻辑里没有这个需求就不用加。

四、总结

功能概括：

  • 对 Chr1/Chr2/Chr3 三个 SV-FASTQ 文件，逐个执行：
    1. map-ont 初次比对 → 提取未比对 reads；
    2. 对 R1 未比对 reads 用 asm20 再比对；
    3. 对 R2 未比对 reads 用自定义 map-ont 参数再比对；
    4. 每一轮都生成 sorted BAM 和 unmapped BAM + unmapped FASTQ；
    5. 只使用 --cs=long，不再使用 --MD。

是否能正常运行：

  • 从 Bash 语法、变量使用、流程控制上看，这段脚本是正确的；
  • 满足前提（文件路径和软件存在）的情况下，可以正常按你的设计一次性跑完三个 Chr。

如果你愿意，我可以在这个版本基础上再加一个“如果某一轮完全匹配就跳过后面轮次”的逻辑，或者帮你加上那个 Col-coty-sup-ChrX-SV-cs.sam 的最终输出。

问题

什么叫某一轮完全匹配就跳过后面轮次

思考

Clarifying the concept of skipping rounds in a mapping pipeline

The phrase "某一轮完全匹配就跳过后面轮次" means skipping subsequent rounds in a pipeline when there are no unmapped reads remaining in a current round. It's efficient

Thought for 5s

回答

我刚才说的“某一轮完全匹配就跳过后面轮次”，其实是一个可选优化思路，不是你脚本现在必须做的事。具体意思是：

1. 现在脚本的逻辑是怎样的？

对每个 Chr，你现在是：

  1. R1：map-ont

    • 输入：原始 FASTQ
    • 输出：
      • 比对好的 R1_SORTED_BAM
      • 未比对的 reads → R1_UNMAPPED_FASTQ

  2. R2：asm20

    • 输入：R1_UNMAPPED_FASTQ（只含 R1 里没比上的 reads）
    • 输出：
      • 比对好的 R2_SORTED_BAM
      • 未比对的 reads → R2_UNMAPPED_FASTQ

  3. R3：planA map-ont（调参）

    • 输入：R2_UNMAPPED_FASTQ
    • 输出：
      • 比对好的 R3_SORTED_BAM
      • 最终三轮都没比上的 reads → R3_UNMAPPED_FASTQ

不管每一轮有多少未比对 reads，脚本都会继续跑到下一轮。

2. “某一轮完全匹配”的意思是什么？

举例说 R1：

  • 在 R1 结束时，你会统计所有未比对 reads 的 ID 并存到
    R1_UNMAPPED_IDS_CLEAN
  • 如果所有 reads 都比对上了，那这一步得到的文件应该是：
bash
R1_UNMAPPED_IDS_CLEAN    # 文件存在但大小为 0（空文件）

也就是说：

这一轮里，已经没有任何未比对的 reads 了。

那这种情况，我就称之为：

“R1 完全匹配”，也可以说“R1 已经把所有 reads 都对上了”。

同理，如果 R2 的未比对 ID 文件 R2_UNMAPPED_IDS_CLEAN 也完全空了，就可以说：

“R2 完全匹配”。

3. 为什么“完全匹配”后可以考虑跳过后续轮次？

假设 R1 就已经全部匹配：

  • R1_UNMAPPED_IDS_CLEAN 是空的
  • R1_UNMAPPED_FASTQ 里也就没有 reads 或者是 0 条

这时继续往下做：

  • R2 输入的是一个没有 reads 的 FASTQ
  • R3 输入的也是从 R2 输出的“0 条 reads FASTQ”

结果：

  • minimap2 会在一个空 FASTQ 上跑，几乎什么都做不了，只是生成一堆“空结果”的 SAM/BAM；
  • 逻辑上没错，但没有实际意义，浪费时间和 I/O。

所以，优化思路是：

  • 如果发现 R1 没有任何未比对 reads：
    • 就说明所有 reads 已经在 R1 这个参数组合下“对上了”；
    • 那么你可以不必再跑 R2 和 R3，直接结束这个 Chr 的流程。

同理，如果：

  • R1 还有部分未比对 reads，就继续 R2；
  • R2 之后如果全都对上了（R2_UNMAPPED_IDS_CLEAN 为空）：
    • 就可以只跑到 R2 为止，不用再做 R3。

4. 如何在你现在的脚本里加这个逻辑？（可选）

注意：这只是“锦上添花”的优化，你现在脚本不加也能正常跑。

以 R1 为例，在 process_fastq 里的这几行后面：

bash
tr -d '\r' < "${R1_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R1_UNMAPPED_IDS_CLEAN}"

echo "[R1] 未比对 ID 行数：$(wc -l < "${R1_UNMAPPED_IDS_CLEAN}")"

可以加一段判断：

bash
# 如果这一轮已经没有未比对 reads，就不用跑 R2/R3 了
if [[ ! -s "${R1_UNMAPPED_IDS_CLEAN}" ]]; then
    echo "[R1] 所有 reads 在 R1 中都已比对上，跳过 R2/R3。"

    # 这里仍然可以保留 R1 的统计结果
    "${SEQKIT}" stats "${IN_FASTQ}" || true

    # 不再进行后续轮次，直接结束这个染色体的处理
    return 0
fi

类似地，在 R2 结束后也可以加：

bash
tr -d '\r' < "${R2_UNMAPPED_IDS}" | awk 'length($0)>0' | LC_ALL=C sort -u > "${R2_UNMAPPED_IDS_CLEAN}"

echo "[R2] 未比对 ID 行数：$(wc -l < "${R2_UNMAPPED_IDS_CLEAN}")"

if [[ ! -s "${R2_UNMAPPED_IDS_CLEAN}" ]]; then
    echo "[R2] 所有剩余 reads 在 R2 中都已比对上，跳过 R3。"
    "${SEQKIT}" stats "${R1_UNMAPPED_FASTQ}" || true
    return 0
fi

这样逻辑就是：

  • 有未比对 reads → 进入下一轮
  • 没有未比对 reads → 这条 Chr 提前结束，不做多余的计算。

5. 总结一句话

“某一轮完全匹配就跳过后面轮次”
就是：如果某一轮比对之后，已经没有任何未比对的 reads（未比对 ID 文件是空的），那再继续往后跑就没有意义，可以直接结束这条染色体的分析，节省时间和资源。

你现在的脚本没有实现这个“跳过”的优化，而是无论如何都会跑完 R1→R2→R3 三轮——这是完全没问题的，只是稍微多用点算力而已。

分享这个问答