trinity运行原理及常见报错（一）

trinity运行过程

1.检测所用软件及输入文件

1)检测输入文件是否正确

Left read files: $VAR1 = [

          '/lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L1_1.fq',

          '/lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L2_1.fq'

        ];

Right read files: $VAR1 = [

          '/lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L1_2.fq',

          '/lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L2_2.fq'

        ];

如果文件不存在或者填写参数时逗号或空格隔开方式不对，报错：

Error, cannot locate file: /lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L1_1.fq,/lustre/02.work/wanglihui/project/Populus_tremula_170417/clean_data/KD_L2_1.fq at /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110//Trinity.pl line
1762.
main::create_full_path(ARRAY(0xe985d8), 1) called at /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110//Trinity.pl line 933

这个报错就是因为旧版本需要reads直接用空格隔开，但是shell中采用了逗号

2)检测各种软件是否正常

需要使用到bowtie samtools java 

旧版本：

Paired mode requires bowtie. Found bowtie at: /lustre/02.work/liuxiaoshuang/biosoft/bowtie/1.1.1/bowtie

Found samtools at: /lustre/00.tools/Bins/samtools

java版本报错：

Error, Trinity requires access to Java version 1.6 or 1.7.  Currently installed version is: java version "1.8.0_91"

Java(TM) SE Runtime Environment (build 1.8.0_91-b14)

Java HotSpot(TM) 64-Bit Server VM (build 25.91-b14, mixed mode)

但是如果分步运行，加上--no_run_butterfly --no_run_quantifygraph 就不会报错，原因可能在于：Inchworm和Chrysalis是C++编写的，而 使用Butterfly是Java编写的，如果运行Butterfly需要检测java可用性

新版本：

Monday, May 15, 2017: 22:15:39 CMD: java -Xmx64m -XX:ParallelGCThreads=5 -jar /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/ExitTester.jar 0

Monday, May 15, 2017: 22:15:43 CMD: java -Xmx64m -XX:ParallelGCThreads=5 -jar /lustre/02.work/liufei/tools/trinityrnaseq-Trinity-v2.4.0/util/support_scripts/ExitTester.jar 1

3)检测trinity版本号

Trinity version: Trinity-v2.4.0

-ERROR: couldn't run the network check to confirm latest Trinity software version.

这一步报错对组装没有影响，也可以调用--no_version_check这个参数，就不显示报错

2.如果上述没有报错，从fq到fa转化

运行命令如下：

Tuesday, May 23, 2017: 10:03:01 CMD: mkdir -p /lustre/02.work/liufei/project/Noref/20170517/Analysis/Basic_Analysis/Assembly/Trinity_assembly/All_Combination/All_Combination_Trinity/chrysalis

CMD finished (0 seconds)

Converting input files. (in parallel)Tuesday, May 23, 2017: 10:03:02
CMD: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /lustre/02.work/liufei/project/Noref/20170517/data/S1/S1_S1_L001_R1_001.fastq >> left.fa

Tuesday, May 23, 2017: 10:03:02 CMD: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /lustre/02.work/liufei/project/Noref/20170517/data/S1/S1_S1_L001_R2_001.fastq
>> right.fa

CMD finished (506 seconds)

Tuesday, May 23, 2017: 10:11:28 CMD: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /lustre/02.work/liufei/project/Noref/20170517/data/S1/S1_S1_L002_R1_001.fastq
>> left.fa

CMD finished (900 seconds)

Tuesday, May 23, 2017: 10:18:02 CMD: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /lustre/02.work/liufei/project/Noref/20170517/data/S1/S1_S1_L002_R2_001.fastq
>> right.fa

CMD finished (484 seconds)

-conversion of 27714705 from FQ to FA format succeeded.

-conversion of 27712988 from FQ to FA format succeeded.

Thursday, December 15, 2016: 11:59:35 CMD: touch left.fa.ok right.fa.ok

Thursday, December 15, 2016: 11:59:35 CMD: cat left.fa right.fa > both.fa

Thursday, December 15, 2016: 12:21:15 CMD: touch both.fa.ok

得到left.fa right.fa both.fa

注：这一步主要是由fq到fa，也可以作为一个软件用于fq2fa

/lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/fastool/fastool --illumina-trinity --to-fasta /lustre/02.work/liufei/test/fq_len_filter/test.fq >> out.fa

3.运行Jellyfish 

building a k-mer catalog from reads

运行命令：

* Running CMD: /data/tools/trinityrnaseq-2.2.0/trinity-plugins/jellyfish/bin/jellyfish count -t 20 -m 25 -s 3726277687  both.fa

* Running CMD: /data/tools/trinityrnaseq-2.2.0/trinity-plugins/jellyfish/bin/jellyfish dump -L 2 mer_counts.jf > jellyfish.kmers.fa

#-L参数是总参数设定的 min_kmer_cov

* Running CMD: /data/tools/trinityrnaseq-2.2.0/trinity-plugins/jellyfish/bin/jellyfish histo -t 20 -o jellyfish.kmers.fa.histo mer_counts.jf

#对kmer的频率做了一个统计

如果内存不足会报错：将参数内存调大即可。

Tuesday, May 23, 2017: 12:25:21 CMD: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/jellyfish/bin/jellyfish count -t 5 -m 25 -s 5450651468  --both-strands  both.fa

Error, cmd: /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110/trinity-plugins/jellyfish/bin/jellyfish count -t 5 -m 25 -s 5450651468  --both-strands  both.fa died with ret 135 at /lustre/02.work/liuxiaoshuang/biosoft/trinity/r20131110//Trinity.pl line
1793.

这一步，主要是-s 这个参数起作用，如果过小会导致报错

主程序中这个参数值是如此定义：my $jelly_hash_size = int( ($max_memory - $read_file_size)/7); # decided upon by Rick Westerman

其中$max_memory是JM设定值，$read_file_size

得到jellyfish.kmers.fa 这是一个kmer库 长度为25