trinity运行原理及常见报错（三）

5.运行Chrysalis

Contig Clustering & de Bruijn Graph Construction

简单介绍：

将上一步生成的contigs聚类，然后对每个component构建Bruijn图。 C++ 

对前一步产生的conting进行聚类：如果conting间有overlap，认为是一类

聚类以后，每个conting集合叫做一个component，对每个component构建De Bruijin graphs

用reads对比，看每一个component支持情况

具体步骤：

1）把可能存在可变剪切及平行基因的conting进行聚类

聚类条件：

如果contings间有k-1(25-1=24)个overlap

如果两个conting上不同位置分别有(k-1)/2个overlap

2)每一个contings集合定义成一个components，每一个components都会构建一个De Bruijin graph 

3)用reads进行回比，看每个component的reads支持情况

命令：

聚类：

inchworm_target: /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa

bowite_reads_fa: /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa

chrysalis_reads_fa: /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa

对inchworm.K25.L25.DS.fa进行初筛：

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/filter_iworm_by_min_length_or_cov.pl /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/inchworm.K25.L25.DS.fa 100 10 > /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/inchworm.K25.L25.DS.fa.min100

建立index

* Running CMD: bowtie2-build -o 3 /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/inchworm.K25.L25.DS.fa.min100 /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/inchworm.K25.L25.DS.fa.min100 1>/dev/null

bowtie2进行比对 得到bam文件 iworm.bowtie.nameSorted.bam

* Running CMD: bash -c " set -o pipefail;bowtie2 --local -k 2 --threads 5 -f --score-min G,46,0 -x /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/inchworm.K25.L25.DS.fa.min100 /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa
 | samtools view -@ 5 -F4 -Sb - | samtools sort -m 5261334937 -@ 5 -no - - > /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm.bowtie.nameSorted.bam" 

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/scaffold_iworm_contigs.pl /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm.bowtie.nameSorted.bam /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/inchworm.K25.L25.DS.fa
> /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm_scaffolds.txt

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/Chrysalis/GraphFromFasta -i /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/inchworm.K25.L25.DS.fa -r /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa
-min_contig_length 200 -min_glue 2 -glue_factor 0.05 -min_iso_ratio 0.05 -t 5 -k 24 -kk 48  -scaffolding /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm_scaffolds.txt  > /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm_cluster_welds_graph.txt

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/Chrysalis/BubbleUpClustering -i /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/inchworm.K25.L25.DS.fa  -weld_graph /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/iworm_cluster_welds_graph.txt
-min_contig_length 200  > /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/GraphFromIwormFasta.out

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/Chrysalis/CreateIwormFastaBundle -i /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/GraphFromIwormFasta.out -o /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/bundled_iworm_contigs.fasta
-min 200

* Running CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/Chrysalis/ReadsToTranscripts -i /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/both.fa -f /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/bundled_iworm_contigs.fasta
-o /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/readsToComponents.out -t 5 -max_mem_reads 50000000 

* Running CMD: /bin/sort -T . -S 49G -k 1,1n /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/readsToComponents.out > /lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/chrysalis/readsToComponents.out.sort

Wednesday, May 17, 2017: 02:09:15 CMD: mkdir -p read_partitions/Fb_0/CBin_0

Wednesday, May 17, 2017: 02:09:15 CMD: mkdir -p read_partitions/Fb_0/CBin_1

Wednesday, May 17, 2017: 02:09:15 CMD: mkdir -p read_partitions/Fb_0/CBin_2

Wednesday, May 17, 2017: 02:09:16 CMD: mkdir -p read_partitions/Fb_0/CBin_3

...

...

...

...

Wednesday, May 17, 2017: 02:14:22 CMD: mkdir -p read_partitions/Fb_0/CBin_487

Wednesday, May 17, 2017: 02:14:22 CMD: mkdir -p read_partitions/Fb_0/CBin_488

Wednesday, May 17, 2017: 02:14:22 CMD: touch partitioned_reads.files.list.ok

Wednesday, May 17, 2017: 02:14:22 CMD: /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file partitioned_reads.files.list --CPU 1 --max_memory
1G  --run_as_paired  --seqType fa --trinity_complete --no_cleanup  --min_kmer_cov  1  --bflyHeapSpaceMax  20G  --bflyGCThreads  5  > recursive_trinity.cmds

Wednesday, May 17, 2017: 02:14:22 CMD: touch recursive_trinity.cmds.ok

Wednesday, May 17, 2017: 02:14:23 CMD: touch recursive_trinity.cmds.ok

得到recursive_trinity.cmds

得到/lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/read_partitions/Fb_0/*/*.trinity.reads.fa

这些output结果将会作为buttrtfly的输入

recursive_trinity.cmds举例：

/lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/../../Trinity --single "/lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/read_partitions/Fb_0/CBin_0/c0.trinity.reads.fa" --output "/lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/read_partitions/Fb_0/CBin_0/c0.trinity.reads.fa.out"
--CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --no_cleanup --min_kmer_cov 1 --bflyHeapSpaceMax 20G --bflyGCThreads 5

/lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/../../Trinity --single "/lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/read_partitions/Fb_0/CBin_0/c1.trinity.reads.fa" --output "/lustre/02.work/liufei/denovo/trinity_test/All_Combination_Trinity/read_partitions/Fb_0/CBin_0/c1.trinity.reads.fa.out"
--CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --no_cleanup --min_kmer_cov 1 --bflyHeapSpaceMax 20G --bflyGCThreads 5
应该是构建graph得到的reads

下一个博客介绍butterfly