使用AmpliconSuite-pipeline进行ecDNA分析

Posted on by KevinZhou

官方github网址：https://github.com/AmpliconSuite/AmpliconSuite-pipeline
申请mosek个人免费学术许可：https://www.mosek.com/products/academic-licenses/
申请后放在~/mosek/下

1. 介绍

Focal oncogene amplification and rearrangements drive tumor growth and evolution in multiple cancer types. Proposed mechanisms for focal amplification include extrachromosomal DNA (ecDNA) formation, breakage-fusion-bridge (BFB) mechanism, tandem duplications, chromothripsis and others. Focally amplified regions are often hotspots for genomic rearrangements. As a result, the focally amplified region may undergo rapid copy number changes and the structure of the focally amplified region may evolve over time contributing to tumor evolution. Furthermore, ecDNA elements may reintegrate back into the genome to form HSRs. The inter-cell heterogeneity in copy number of ecDNA as well as the interchangeability between ecDNA and HSR may allow the tumor to adapt to changing environment, e.g. targetted drug application. As a result, understanding the architecture of the focal amplifications is important to gain insights into cancer biology. AmpliconArchitect (AA) is a tool which can reconstruct the structure of focally amplified regions (>10kbp) in a cancer sample using whole genome sequence short paired-end data.

2. 安装流程：

conda create -n ampsuite && conda activate ampsuite
conda install -c bioconda -c conda-forge ampliconsuite 
conda install -c mosek mosek

# then run the installer script to finalize the locations of the data repo and mosek license 
wget https://raw.githubusercontent.com/AmpliconSuite/AmpliconSuite-pipeline/master/install.sh
chmod +x install.sh
./install.sh --finalize_only  # -h to see options

额外自行下载数据库: https://refs.ampliconrepository.org/?prefix=data/module_support_files/AmpliconArchitect/
已经经过bwa比对，下载没有index的GRCh38.tar.gz即可

3. 并行脚本：

AA_DATA_REPO=~/database/AmpliconArchitect_repo/

perl -ne 'chomp; next if /^$/; @a = split /\t/; print "AmpliconSuite-pipeline.py -s $a[0] -t 4 --normal_bam ../align/$a[1]_bqsr.bam --bam ../align/$a[0]_bqsr.bam --run_AA --run_AC > log/$a[0].log 2>&1 && echo $a[0] AmpliconSuite ok\n";' ../mutect2/sample_pair.txt > RunAmpliconSuite.sh

# 输出示例：AmpliconSuite-pipeline.py -s FETB06-BLPT-E -t 4 --normal_bam ../align/FETB06-N_bqsr.bam --bam ../align/FETB06-BLPT-E_bqsr.bam --run_AA --run_AC > log/FETB06-BLPT-E.log 2>&1 && echo FETB06-BLPT-E AmpliconSuite ok

nohup bash -c "cat RunAmpliconSuite.sh | parallel -j 4" > log/AmpliconSuite.log 2>&1 &

4. 输出结果解读:

AmpliconArchitect 最终输出结果为：SAMPLE_AA_results/SAMPLE_summary.txt，其中包含了“局部扩增区域”

#Amplicons = 8
-----------------------------------------------------------------------------------------
[amplicon1] AmpliconID = 1
[amplicon1] #Intervals = 1
[amplicon1] Intervals = chr2:94499495-94573455
[amplicon1] OncogenesAmplified = ,
[amplicon1] TotalIntervalSize = 73961
[amplicon1] AmplifiedIntervalSize = 73960
[amplicon1] AverageAmplifiedCopyCount = 6.88546257606703
[amplicon1] #Chromosomes = 1
[amplicon1] #SequenceEdges = 1
[amplicon1] #BreakpointEdges = 0
[amplicon1] #CoverageShifts = 0
[amplicon1] #MeanshiftSegmentsCopyCount>5 = 0
[amplicon1] #Foldbacks = 0
[amplicon1] #CoverageShiftsWithBreakpointEdges = 0
[amplicon1] #TotalAmpliconWeight = 509255.698
[amplicon1] #FractionWeightInDecomp = 1.00000000
[amplicon1] -----------------------------------------------------------------------------------------
[amplicon2] AmpliconID = 2
[amplicon2] #Intervals = 1
[amplicon2] Intervals = chr3:90315297-90505827
[amplicon2] OncogenesAmplified = ,
[amplicon2] TotalIntervalSize = 190531
[amplicon2] AmplifiedIntervalSize = 190528
[amplicon2] AverageAmplifiedCopyCount = 5.204942372970038
[amplicon2] #Chromosomes = 1
[amplicon2] #SequenceEdges = 3
[amplicon2] #BreakpointEdges = 1
[amplicon2] #CoverageShifts = 0
[amplicon2] #MeanshiftSegmentsCopyCount>5 = 0
[amplicon2] #Foldbacks = 0
[amplicon2] #CoverageShiftsWithBreakpointEdges = 0
[amplicon2] #TotalAmpliconWeight = 991702.875
[amplicon2] #FractionWeightInDecomp = 1.00001048
[amplicon2] -----------------------------------------------------------------------------------------

AmpliconClassifier 最终输出结果为：

  1. SAMPLE_amplicon_classification_profiles.tsv
  2. SAMPLE_gene_list.tsv
  3. SAMPLE_ecDNA_context_calls.tsv

SAMPLE_amplicon_classification_profiles.tsv:

sample_name     amplicon_number amplicon_decomposition_class    ecDNA+  BFB+    ecDNA_amplicons
FETB06-BLPT-M   amplicon1       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon2       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon3       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon4       Complex-non-cyclic      None detected   None detected   0
FETB06-BLPT-M   amplicon5       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon6       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon7       Linear  None detected   None detected   0
FETB06-BLPT-M   amplicon8       Linear  None detected   None detected   0

合并文件：

find . -path './*_classification/*_amplicon_classification_profiles.tsv' \
  | sort \
  | xargs awk '
FNR==1 {
    if (NR==1) print $0
    next
}
{
    print $0
}
' > All_amplicon_classification_profiles.tsv