使用IsoSeq3利用pacbio測序數據進行全長轉錄本序列分析，為了得到更準確的基因注釋作準備，但價格昂貴，且無法進行表達量分析

mkdir -p /home/train/08.RNA-seq_analysis_by_trinity
cd /home/train/08.RNA-seq_analysis_by_trinity

# 對PacBio轉錄組測序數據進行全長轉錄本序列分析
# https://isoseq.how/clustering/schematic-workflow.html
mkdir -p /home/train/08.RNA-seq_analysis_by_trinity/IsoSeq
cd /home/train/08.RNA-seq_analysis_by_trinity/IsoSeq

#將subreads轉換成ccs數據
PATH=/opt/biosoft/miniconda3_for_pbbioconda/bin/:$PATH
samtools view -h ~/00.incipient_data/data_for_gene_prediction_and_RNA-seq/m54086_170204_081430.subreads.bam | head -n 10000 | samtools view -b > m54086_170204_081430.subreads.bam
pbindex m54086_170204_081430.subreads.bam #對pacbio測序結果的bam建立索引
# ccs的正常運行需要提供chemistry.xml測序試劑信息;在運行ccs數據之前配置文件chemistry.xml 
# wget https://raw.githubusercontent.com/PacificBiosciences/pbcore/develop/pbcore/chemistry/resources/mapping.xml -O chemistry.xml
cp ~/00.incipient_data/data_for_genome_assembling/chemistry.xml .
perl -p -i -e 's/<SoftwareVersion>5.0/<SoftwareVersion>4.0/' chemistry.xml
export SMRT_CHEMISTRY_BUNDLE_DIR=$PWD#將當前目錄寫到環境變量
# 運行ccs命令將Subreads轉換為CCS數據。默認參數--min-passes為3表示至少有3條全長的subreads；--min-snr為2.5表示信噪比，即SSubreads的準確率至少為2.5/3.5=71.4%。
ccs --min-rq 0.9 m54086_170204_081430.subreads.bam m54086_170204_081430.ccs.bam
#使用samtools對結果進行查看
[train@MiWiFi-R3P-srv IsoSeq]$ samtools view m54086_170204_081430.ccs.bam | les

# 去除引物和barcode堿基信息。
echo '>NEB_5p
GCAATGAAGTCGCAGGGTTGGG
>Clontech_5p
AAGCAGTGGTATCAACGCAGAGTACATGGGG
>NEB_Clontech_3p
GTACTCTGCGTTGATACCACTGCTT' > barcoded_primers.fasta
lima m54086_170204_081430.ccs.bam barcoded_primers.fasta m54086_170204_081430.lima.bam --isoseq --peek-guess

統計篩選后的條數
[train@MiWiFi-R3P-srv IsoSeq]$ samtools view m54086_170204_081430.lima.Clontech_5p--NEB_Clontech_3p.bam | wc -l
656


# 去除PolyA和合體序列，得到FLNC序列
isoseq refine m54086_170204_081430*lima*.bam barcoded_primers.fasta m54086_170204_081430.flnc.bam --require-polya

# 對FLNC reads進行聚類，得到全長轉錄本序列信息: hq.fasta.gz with predicted accuracy ≥ 0.99; lq.fasta.gz with predicted accuracy < 0.99。
isoseq cluster m54086_170204_081430.flnc.bam clustered.bam --verbose --use-qvs

[train@MiWiFi-R3P-srv IsoSeq]$ samtools view m54086_170204_081430.flnc.bam | wc -l
646
[train@MiWiFi-R3P-srv IsoSeq]$ samtools view clustered.bam | wc -l
41
[train@MiWiFi-R3P-srv IsoSeq]$ ll -t
total 37784
-rw-r--r-- 1 train train     8588 Jul 21 14:36 clustered.cluster
-rw-r--r-- 1 train train    15327 Jul 21 14:36 clustered.hq.fasta.gz
-rw-r--r-- 1 train train     2752 Jul 21 14:36 clustered.lq.fasta.gz
-rw-r--r-- 1 train train      404 Jul 21 14:36 clustered.bam.pbi
-rw-r--r-- 1 train train    21560 Jul 21 14:36 clustered.bam
-rw-r--r-- 1 train train      381 Jul 21 14:36 clustered.hq.bam.pbi
-rw-r--r-- 1 train train    18707 Jul 21 14:36 clustered.hq.bam
-rw-r--r-- 1 train train      109 Jul 21 14:36 clustered.lq.bam.pbi
-rw-r--r-- 1 train train     3352 Jul 21 14:36 clustered.lq.bam
-rw-r--r-- 1 train train     1568 Jul 21 14:36 clustered.transcriptset.xml
-rw-r--r-- 1 train train     6505 Jul 21 14:36 clustered.cluster_report.csv
-rw-r--r-- 1 train train     8195 Jul 21 14:33 m54086_170204_081430.flnc.bam.pbi
-rw-r--r-- 1 train train  1509378 Jul 21 14:33 m54086_170204_081430.flnc.bam
-rw-r--r-- 1 train train     1611 Jul 21 14:33 m54086_170204_081430.flnc.consensusreadset.xml
-rw-r--r-- 1 train train      821 Jul 21 14:33 m54086_170204_081430.flnc.filter_summary.report.json
-rw-r--r-- 1 train train    50339 Jul 21 14:33 m54086_170204_081430.flnc.report.csv
-rw-r--r-- 1 train train     1990 Jul 21 14:29 m54086_170204_081430.lima.Clontech_5p--NEB_Clontech_3p.consensusreadset.xml
-rw-r--r-- 1 train train     3826 Jul 21 14:29 m54086_170204_081430.lima.consensusreadset.xml
-rw-r--r-- 1 train train      639 Jul 21 14:29 m54086_170204_081430.lima.json
-rw-r--r-- 1 train train      108 Jul 21 14:29 m54086_170204_081430.lima.lima.counts
-rw-r--r-- 1 train train      845 Jul 21 14:29 m54086_170204_081430.lima.lima.summary
-rw-r--r-- 1 train train     8342 Jul 21 14:29 m54086_170204_081430.lima.Clontech_5p--NEB_Clontech_3p.bam.pbi
-rw-r--r-- 1 train train  1564566 Jul 21 14:29 m54086_170204_081430.lima.Clontech_5p--NEB_Clontech_3p.bam
-rw-r--r-- 1 train train   101927 Jul 21 14:29 m54086_170204_081430.lima.lima.clips
-rw-r--r-- 1 train train   183452 Jul 21 14:29 m54086_170204_081430.lima.lima.report
-rw-r--r-- 1 train train      118 Jul 21 14:29 m54086_170204_081430.lima.lima.guess
-rw-r--r-- 1 train train      119 Jul 21 14:29 barcoded_primers.fasta
-rw-r--r-- 1 train train      862 Jul 21 14:17 m54086_170204_081430.ccs.ccs_report.txt
-rw-r--r-- 1 train train    61814 Jul 21 14:17 m54086_170204_081430.ccs.zmw_metrics.json.gz
-rw-r--r-- 1 train train     8030 Jul 21 14:17 m54086_170204_081430.ccs.bam.pbi
-rw-r--r-- 1 train train  1694358 Jul 21 14:17 m54086_170204_081430.ccs.bam
-rw-r--r-- 1 train train     8366 Jul 21 14:15 chemistry.xml
-rw-r--r-- 1 train train    89364 Jul 21 14:10 m54086_170204_081430.subreads.bam.pbi
-rw-r--r-- 1 train train 33228366 Jul 21 14:09 m54086_170204_081430.subreads.bam