【5.4.1】Nanopore_psU

一、安装

git clone https://github.com/sihaohuanguc/Nanopore_psU.git  
pip install .
nanopsu -h

二、使用

1.先使用guppy来call， 生成fastq文件

guppy_basecaller --input_path fast5 \
				 --recursive \
				 --save_path fastq \
				 --records_per_fastq 0 \
				 --flowcell FLO-MIN106 \
				 --kit SQK-RNA002 \
				 --qscore_filtering \
				 --min_qscore 7 \
				 --cpu_threads_per_caller 3 \
				 --num_callers 5

2.Alignment and pile up

nanopsu alignment -i path/of/fastq/ -r reference.fa

生成的文件

$ ls alignment/plus_strand/
collect.bam    collect_pile.txt  collect.sorted.bam  reference.fa.fai
collect.fastq  collect.sam       reference.fa

3.特征提取

删掉未匹配的位点，用>符号表示的

nanopsu remove_intron

某个位置的U，至少有20个reads覆盖，才进一步的提取

nanopsu extract_features

4.psU预测

nanopsu prediction

三、文献解读

纳米孔数据预处理

1. 在测序过程中生成的所有原始 fast5 文件，由 guppy 碱基调用程序（版本 3.2.2+9fe0a78）使用 min_qscore 7 调用的碱基。
2. 由 minimap2（版本 2.18-r1015）[ 37 ] 与参数 -ax splice -uf -k14 对齐。 (这样的比对，的确会得到更多的序列)
3. “错误”特征是通过定制的 Python 脚本（ https://github.com/sihaohuanguc/Nanopore_psU ）从 mpileup 文件中提取的。

四、报错

4.1 报错

No module name 'sklearn.ensemble.forest'  

解决方法：

pip install scikit-learn==0.23.2 --timeout=100000

4.2 报错

 undefined reference to `lzma_end'

解决：

dnf install xz-libs


g++ -o nanopolish -g -O3 -std=c++11 -fopenmp -fsigned-char -D_FILE_OFFSET_BITS=64  -I./include -I./htslib -I./minimap2 -I./src -I./src/hmm -I./src/thirdparty -I./src/thirdparty/scrappie -I./src/common -I./src/alignment -I./src/pore_model -I./src/io -I./src/basemods -I./eigen/ -I./slow5lib/include/ -fPIC src/main/nanopolish.o src/nanopolish_variant_db.o src/nanopolish_vcf2fasta.o src/nanopolish_polya_estimator.o src/nanopolish_call_methylation.o src/nanopolish_haplotype.o src/nanopolish_squiggle_read.o src/nanopolish_detect_polyi.o src/nanopolish_read_db.o src/nanopolish_methyltrain.o src/nanopolish_raw_loader.o src/nanopolish_index.o src/nanopolish_scorereads.o src/training_core.o src/nanopolish_phase_reads.o src/nanopolish_fast5_check.o src/nanopolish_call_variants.o src/nanopolish_train_poremodel_from_basecalls.o src/hmm/nanopolish_duration_model.o src/hmm/nanopolish_profile_hmm.o src/hmm/nanopolish_profile_hmm_r9.o src/hmm/nanopolish_transition_parameters.o src/hmm/nanopolish_profile_hmm_r7.o src/common/fs_support.o src/common/nanopolish_bam_processor.o src/common/nanopolish_alphabet.o src/common/logsum.o src/common/nanopolish_iupac.o src/common/nanopolish_klcs.o src/common/nanopolish_common.o src/common/nanopolish_variant.o src/common/nanopolish_bam_utils.o src/alignment/nanopolish_eventalign.o src/alignment/nanopolish_anchor.o src/alignment/nanopolish_alignment_db.o src/pore_model/nanopolish_model_names.o src/pore_model/nanopolish_pore_model_set.o src/pore_model/nanopolish_poremodel.o src/io/nanopolish_fast5_processor.o src/io/nanopolish_fast5_loader.o src/io/nanopolish_fast5_io.o src/basemods/nanopolish_basemods.o src/thirdparty/fet.o src/thirdparty/stdaln.o src/thirdparty/scrappie/event_detection.o src/thirdparty/scrappie/scrappie_common.o ./htslib/libhts.a ./minimap2/libminimap2.a ./slow5lib/lib/libslow5.a ./lib/libhdf5.a -lz -lrt -ldl

./htslib/libhts.a(hts.o): In function `decompress_peek_xz':
/data/software/htslib/hts.c:347: undefined reference to `lzma_easy_decoder_memusage'
/data/software/htslib/hts.c:347: undefined reference to `lzma_stream_decoder'
/data/software/htslib/hts.c:355: undefined reference to `lzma_code'
/data/software/htslib/hts.c:362: undefined reference to `lzma_end'
/data/software/htslib/hts.c:357: undefined reference to `lzma_end'
./htslib/libhts.a(cram_io.o): In function `cram_compress_by_method':
/data/software/nanopolish/htslib/cram/cram_io.c:1776: undefined reference to `BZ2_bzBuffToBuffCompress'
./htslib/libhts.a(cram_io.o): In function `lzma_mem_deflate':
/data/software/nanopolish/htslib/cram/cram_io.c:1293: undefined reference to `lzma_stream_buffer_bound'
/data/software/nanopolish/htslib/cram/cram_io.c:1299: undefined reference to `lzma_easy_buffer_encode'
./htslib/libhts.a(cram_io.o): In function `cram_uncompress_block':
/data/software/nanopolish/htslib/cram/cram_io.c:1612: undefined reference to `BZ2_bzBuffToBuffDecompress'
./htslib/libhts.a(cram_io.o): In function `lzma_mem_inflate':
/data/software/nanopolish/htslib/cram/cram_io.c:1315: undefined reference to `lzma_easy_decoder_memusage'
/data/software/nanopolish/htslib/cram/cram_io.c:1315: undefined reference to `lzma_stream_decoder'
/data/software/nanopolish/htslib/cram/cram_io.c:1333: undefined reference to `lzma_code'
/data/software/nanopolish/htslib/cram/cram_io.c:1346: undefined reference to `lzma_code'
/data/software/nanopolish/htslib/cram/cram_io.c:1357: undefined reference to `lzma_end'
/data/software/nanopolish/htslib/cram/cram_io.c:1362: undefined reference to `lzma_end'
/data/software/nanopolish/htslib/cram/cram_io.c:1357: undefined reference to `lzma_end'
./htslib/libhts.a(arith_dynamic.o): In function `arith_compress_to':
/data/software/nanopolish/htslib/htscodecs/htscodecs/arith_dynamic.c:879: undefined reference to `BZ2_bzBuffToBuffCompress'

-llzma -lbz2 添加到末尾

g++ -o nanopolish -g -O3 -std=c++11 -fopenmp -fsigned-char -D_FILE_OFFSET_BITS=64  -I./include -I./htslib -I./minimap2 -I./src -I./src/hmm -I./src/thirdparty -I./src/thirdparty/scrappie -I./src/common -I./src/alignment -I./src/pore_model -I./src/io -I./src/basemods -I./eigen/ -I./slow5lib/include/ -fPIC src/main/nanopolish.o src/nanopolish_variant_db.o src/nanopolish_vcf2fasta.o src/nanopolish_polya_estimator.o src/nanopolish_call_methylation.o src/nanopolish_haplotype.o src/nanopolish_squiggle_read.o src/nanopolish_detect_polyi.o src/nanopolish_read_db.o src/nanopolish_methyltrain.o src/nanopolish_raw_loader.o src/nanopolish_index.o src/nanopolish_scorereads.o src/training_core.o src/nanopolish_phase_reads.o src/nanopolish_fast5_check.o src/nanopolish_call_variants.o src/nanopolish_train_poremodel_from_basecalls.o src/hmm/nanopolish_duration_model.o src/hmm/nanopolish_profile_hmm.o src/hmm/nanopolish_profile_hmm_r9.o src/hmm/nanopolish_transition_parameters.o src/hmm/nanopolish_profile_hmm_r7.o src/common/fs_support.o src/common/nanopolish_bam_processor.o src/common/nanopolish_alphabet.o src/common/logsum.o src/common/nanopolish_iupac.o src/common/nanopolish_klcs.o src/common/nanopolish_common.o src/common/nanopolish_variant.o src/common/nanopolish_bam_utils.o src/alignment/nanopolish_eventalign.o src/alignment/nanopolish_anchor.o src/alignment/nanopolish_alignment_db.o src/pore_model/nanopolish_model_names.o src/pore_model/nanopolish_pore_model_set.o src/pore_model/nanopolish_poremodel.o src/io/nanopolish_fast5_processor.o src/io/nanopolish_fast5_loader.o src/io/nanopolish_fast5_io.o src/basemods/nanopolish_basemods.o src/thirdparty/fet.o src/thirdparty/stdaln.o src/thirdparty/scrappie/event_detection.o src/thirdparty/scrappie/scrappie_common.o ./htslib/libhts.a ./minimap2/libminimap2.a ./slow5lib/lib/libslow5.a ./lib/libhdf5.a -lz -lrt -ldl  -llzma -lbz2

解决办法：

wget -c https://nchc.dl.sourceforge.net/project/bzip2/bzip2-1.0.6.tar.gz
tar -xzvf  bzip2-1.0.6.tar.gz
cd bzip2-1.0.6
make -f Makefile-libbz2_so
make clean
make
make install

参考资料

• https://github.com/sihaohuanguc/Nanopore_psU
• https://genomebiology.biomedcentral.com/articles/10.1186/s13059-021-02557-y
• https://blog.csdn.net/u013010499/article/details/113105907
• https://github.com/vcflib/vcflib/issues/254