fgbio，picard处理带有UMI的fq序列

UMI:unique molecular identifier，UMI建库检测稀有突变、校正测序错误与PCR偏差等。

得到包含UMI分子标签信息的BAM文件

1.提取UMI

# 也能处理.gz 压缩文件

picard FastqToSam F1=test_read1.fq F2=test_read2.fq O=test.uBam SM=testsample

fgbio  -Xmx50G ExtractUmisFromBam  -i  test.uBam  -o  test.umi.uBam  -r  5M2S+T 5M2S+T  -s  RX -t ZA  ZB

2. 比对去掉umi的序列

samtools fastq  test.umi.uBam | bwa mem -t 50 -p /data/ref/hg38/hg38 /dev/stdin | samtools view -b > test.umi.Bam

3. 合并uBam 和 Bam 得到带有UMI信息的比对文件

picard MergeBamAlignment R=/data/ref/hg38/hg38.fa \
    UNMAPPED_BAM=test.umi.uBam  \
    ALIGNED_BAM=test.umi.Bam \
    O=test.umi.merged.Bam  \
    CREATE_INDEX=true    \
    MAX_GAPS=-1 \
    ALIGNER_PROPER_PAIR_FLAGS=true \
    VALIDATION_STRINGENCY=SILENT \
    SO=coordinate \
    ATTRIBUTES_TO_RETAIN=XS

4. Call Consensus Reads

fgbio GroupReadsByUmi \
    --input=test.umi.merged.Bam \
    --output=test.umi.group.Bam  \
    --strategy=paired  --min-map-q=20  --edits=1 --raw-tag=RX

fgbio CallMolecularConsensusReads \
    --min-reads=1 \
    --min-input-base-quality=20 \
    --input=test.umi.group.Bam \
    --output=test.consensus.uBam

samtools fastq test.consensus.uBam | bwa mem -t 50 -p /data/ref/hg38/hg38  /dev/stdin | samtools view -b - > test.consensus.Bam

picard MergeBamAlignment R=/data/ref/hg38/hg38.fa \
    UNMAPPED_BAM=test.consensus.uBam  \
    ALIGNED_BAM=test.consensus.Bam \
    O=test.consensus.merge.Bam  \
    CREATE_INDEX=true    \
    MAX_GAPS=-1 \
    ALIGNER_PROPER_PAIR_FLAGS=true \
    VALIDATION_STRINGENCY=SILENT \
    SO=coordinate \
    ATTRIBUTES_TO_RETAIN=XS

fgbio FilterConsensusReads \
    --input=test.consensus.merge.Bam  \
    --output=test.consensus.merge.filter.Bam \
    --ref=/data/ref/hg38/hg38.fa --min-reads=2 \
    --max-read-error-rate=0.05 \
    --max-base-error-rate=0.1 \
    --min-base-quality=30 \
    --max-no-call-fraction=0.20

fgbio ClipBam \
    --input=test.consensus.merge.filter.Bam   \
    --output=test.consensus.merge.filter.clip.Bam \
    --ref=/data/ref/hg38/hg38.fa  \
    --clip-overlapping-reads=true

参考：

http://fulcrumgenomics.github.io/fgbio/tools/latest/
https://broadinstitute.github.io/picard/command-line-overview.html#LiftOverIntervalList