轉錄組分析實戰附錄:Trinity拼接結果質量控制

2023年12月9日發(作者：犯錯檢討書)

轉錄組分析實戰附錄：Trinity拼接結果質量控制

在第二節中，我們采用了Trinity工具做了轉錄組數據的拼接，我一共是6個樣本6個

G的數據量，在我那個設置下跑了接近30多個小時就完成了拼接工作。

那么今天的工作就是通過RSeQC這個軟件對拼接結果進行一個質量控制與可視化

這個軟件主要是針對于一些臨床RNAq的數據以及有參考基因組的數據，但是對沒

有參考基因組的RNAq數據就很多Tool沒有辦法使用。

首先，通過bowtie2對得到的Trinity拼接好的fasta格式進行構建Index

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ ll | sort -nk 7total 86667684

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:08

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:19 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:24 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:26 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .iworm_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 ed

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 partitioned_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 recursive_

-rw-rw-r-- 1 yeyt yeyt 9 Sep 14 01:08 _count

-rw-rw-r-- 1 yeyt yeyt 10 Sep 14 01:59 _count

-rw-rw-r-- 1 yeyt yeyt 2757 Sep 14 10:31

-rw-rw-r-- 1 yeyt yeyt 22843 Sep 14 01:26

-rw-rw-r-- 1 yeyt yeyt 13366802 Sep 14 10:39 partitioned_

-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 14 10:39 recursive_

-rw-rw-r-- 1 yeyt yeyt 493022300 Sep 14 03:27

-rw-rw-r-- 1 yeyt yeyt Sep 14 01:08

-rw-rw-r-- 1 yeyt yeyt 26501596675 Sep 14 01:24

-rw-rw-r-- 1 yeyt yeyt 49568938526 Sep 14 06:38 scaffolding_

drwxrwxr-x 2 yeyt yeyt 4096 Sep 14 10:33 chrysalis/

drwxrwxr-x 3 yeyt yeyt 4096 Sep 14 01:03 insilico_read_normalization/

drwxrwxr-x 4 yeyt yeyt 4096 Sep 14 10:39 read_partitions/

-rw-rw-r-- 1 yeyt yeyt 0 Sep 15 20:51 align_

-rw-rw-r-- 1 yeyt yeyt 62 Sep 15 20:51

-rw-rw-r-- 1 yeyt yeyt 651 Sep 15 07:03

-rw-rw-r-- 1 yeyt yeyt 10213332 Sep 15 07:03 _trans_map

-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 15 07:03 recursive_ted

-rw-rw-r-- 1 yeyt yeyt 244740565 Sep 15 07:03

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2-build

Settings:

Output files: ".*.bt2"

Line rate: 6 (line is 64 bytes)

Lines per side: 1 (side is 64 bytes)

Offt rate: 4 (one in 16)

FTable chars: 10

Strings: unpacked

Max bucket size: default

Max bucket size, sqrt multiplier: default

Max bucket size, len divisor: 4

Difference-cover sample period: 1024

Endianness: little

Actual local endianness: little

Sanity checking: disabled

Asrtions: disabled

Random ed: 0

Sizeofs: void*:8, int:4, long:8, size_t:8

Input files DNA, FASTA:

Building a SMALL index

Reading reference sizesReading reference sizes Time reading reference sizes: 00:00:03Calculating joined lengthWriting headerRerving space for joined stringJoining Exiting Ebwt::buildToDisk()Returning from initFromVectorWrote 103828770 bytes to primary EBWT file: 2Wrote 55572488 bytes to condary EBWT file: 2Re-opening _in1 and _in2 as input streamsReturning from Ebwt constructorHeaders: len: 222289920 bwtLen: 222289921 sz: 55572480 bwtSz: 55572481 lineRate: 6 offRate: 4 offMask: 0xfffffff0 ftabChars: 10 eftabLen: 20 eftabSz: 80 ftabLen: 1048577 ftabSz: 4194308 offsLen: 13893121 offsSz: 55572484 lineSz: 64 sideSz: 64 sideBwtSz: 48 sideBwtLen: 192 numSides: 1157761 numLines: 1157761 ebwtTotLen: 74096704 ebwtTotSz: 74096704 color: 0 rever: 1yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ ll | sort -nk 7total 86822504-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07 -rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07 -rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:08 -rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:19 .jellyfish_-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:24 .jellyfish_-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:26 .jellyfish_-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .iworm_-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 ed-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 partitioned_-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 recursive_-rw-rw-r-- 1 yeyt yeyt 9 Sep 14 01:08 _count-rw-rw-r-- 1 yeyt yeyt 10 Sep 14 01:59 _count-rw-rw-r-- 1 yeyt yeyt 2757 Sep 14 10:31 -rw-rw-r-- 1 yeyt yeyt 22843 Sep 14 01:26 -rw-rw-r-- 1 yeyt yeyt 13366802 Sep 14 10:39 partitioned_-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 14 10:39 recursive_-rw-rw-r-- 1 yeyt yeyt 493022300 Sep 14 03:27 -rw-rw-r-- 1 yeyt yeyt Sep 14 01:08 -rw-rw-r-- 1 yeyt yeyt 26501596675 Sep 14 01:24 -rw-rw-r-- 1 yeyt yeyt 49568938526 Sep 14 06:38 scaffolding_xrwxr-x 2 yeyt yeyt 4096 Sep 14 10:33 chrysalis/drwxrwxr-x 3 yeyt yeyt 4096 Sep 14 01:03 insilico_read_normalization/drwxrwxr-x 4 yeyt yeyt 4096 Sep 14 10:39 read_partitions/-rw-rw-r-- 1 yeyt yeyt 0 Sep 15 20:51 align_-rw-rw-r-- 1 yeyt yeyt 62 Sep 15 20:51 -rw-rw-r-- 1 yeyt yeyt 651 Sep 15 07:03 -rw-rw-r-- 1 yeyt yeyt 10213332 Sep 15 07:03 _trans_map-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 15 07:03 recursive_ted-rw-rw-r-- 1 yeyt yeyt 244740565 Sep 15 07:03 rwxrwxr-x 3 yeyt yeyt 4096 Sep 15 18:42 ../drwxrwxr-x 5 yeyt yeyt 4096 Sep 15 20:51 ./-rw-rw-r-- 1 yeyt yeyt 1984490 Sep 23 13:50 2-rw-rw-r-- 1 yeyt yeyt 55572480 Sep 23 13:50 2

-rw-rw-r-- 1 yeyt yeyt 55572480 Sep 23 13:50 2

-rw-rw-r-- 1 yeyt yeyt 55572488 Sep 23 14:03 2

-rw-rw-r-- 1 yeyt yeyt 55572488 Sep 23 14:16 2

-rw-rw-r-- 1 yeyt yeyt 103828770 Sep 23 14:03 2

-rw-rw-r-- 1 yeyt yeyt 103828770 Sep 23 14:16 2

在最后生成的6個以bt2結尾的則是Index文件

接下來進行Bowtie2回貼并生成sam文件

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/ass

#最后生成的以下文件log：

#回貼B251的雙端測序結果

perl: warning: Setting locale failed.

perl: warning: Plea check that your locale ttings:

LANGUAGE = "en_US:en",

LC_ALL = (unt),

LC_PAPER = "zh_-8",

LC_ADDRESS = "zh_-8",

LC_MONETARY = "zh_-8",

LC_NUMERIC = "zh_-8",

LC_TELEPHONE = "zh_-8",

LC_IDENTIFICATION = "zh_-8",

LC_MEASUREMENT = "zh_-8",

LC_TIME = "zh_-8",

LC_NAME = "zh_-8",

LANG = "en_-8"

are supported and installed on your system.

perl: warning: Falling back to the standard locale ("C").

28213701 reads; of the:

28213701 (100.00%) were paired; of the:

3865337 (13.70%) aligned concordantly 0 times

2140365 (7.59%) aligned concordantly exactly 1 time

22207999 (78.71%) aligned concordantly >1 times

----

3865337 pairs aligned concordantly 0 times; of the:

134400 (3.48%) aligned discordantly 1 time

----

3730937 pairs aligned 0 times concordantly or discordantly; of the:

7461874 mates make up the pairs; of the:

2553395 (34.22%) aligned 0 times

273693 (3.67%) aligned exactly 1 time

4634786 (62.11%) aligned >1 times

95.47% overall alignment rate

#回貼B252的雙端測序結果

perl: warning: Setting locale failed.

perl: warning: Plea check that your locale ttings:

LANGUAGE = "en_US:en",

LC_ALL = (unt),

LC_PAPER = "zh_-8",

LC_ADDRESS = "zh_-8",

LC_MONETARY = "zh_-8",

LC_NUMERIC = "zh_-8",

LC_TELEPHONE = "zh_-8",

LC_IDENTIFICATION = "zh_-8",

LC_MEASUREMENT = "zh_-8",

LC_TIME = "zh_-8",

LC_NAME = "zh_-8",

LANG = "en_-8"

are supported and installed on your system.

perl: warning: Falling back to the standard locale ("C").

24423445 reads; of the:

24423445 (100.00%) were paired; of the:

2755943 (11.28%) aligned concordantly 0 times

2003579 (8.20%) aligned concordantly exactly 1 time

19663923 (80.51%) aligned concordantly >1 times

----

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/ ---- 2755943 pairs aligned concordantly 0 times; of the: 82738 (3.00%) aligned discordantly 1 time ---- 2673205 pairs aligned 0 times concordantly or discordantly; of the: 5346410 mates make up the pairs; of the: 1943923 (36.36%) aligned 0 times 258490 (4.83%) aligned exactly 1 time 3143997 (58.81%) aligned >1 times96.02% overall alignment rate#回貼R251的雙端測序結果perl: warning: Setting locale : warning: Plea check that your locale ttings: LANGUAGE = "en_US:en", LC_ALL = (unt), LC_PAPER = "zh_-8", LC_ADDRESS = "zh_-8", LC_MONETARY = "zh_-8", LC_NUMERIC = "zh_-8", LC_TELEPHONE = "zh_-8", LC_IDENTIFICATION = "zh_-8", LC_MEASUREMENT = "zh_-8", LC_TIME = "zh_-8", LC_NAME = "zh_-8", LANG = "en_-8" are supported and installed on your : warning: Falling back to the standard locale ("C").24498964 reads; of the: 24498964 (100.00%) were paired; of the: 2605874 (10.64%) aligned concordantly 0 times 2058157 (8.40%) aligned concordantly exactly 1 time 19834933 (80.96%) aligned concordantly >1 times ---- 2605874 pairs aligned concordantly 0 times; of the: 68645 (2.63%) aligned discordantly 1 time ---- 2537229 pairs aligned 0 times concordantly or discordantly; of the: 5074458 mates make up the pairs; of the: 1920173 (37.84%) aligned 0 times 259673 (5.12%) aligned exactly 1 time 2894612 (57.04%) aligned >1 times96.08% overall alignment rate#回貼R252的雙端測序結果perl: warning: Setting locale : warning: Plea check that your locale ttings: LANGUAGE = "en_US:en", LC_ALL = (unt), LC_PAPER = "zh_-8", LC_ADDRESS = "zh_-8", LC_MONETARY = "zh_-8", LC_NUMERIC = "zh_-8", LC_TELEPHONE = "zh_-8", LC_IDENTIFICATION = "zh_-8", LC_MEASUREMENT = "zh_-8", LC_TIME = "zh_-8", LC_NAME = "zh_-8", LANG = "en_-8" are supported and installed on your : warning: Falling back to the standard locale ("C").23929511 reads; of the: 23929511 (100.00%) were paired; of the: 3455581 (14.44%) aligned concordantly 0 times 1770888 (7.40%) aligned concordantly exactly 1 time 18703042 (78.16%) aligned concordantly >1 times ---- 3455581 pairs aligned concordantly 0 times; of the: 132348 (3.83%) aligned discordantly 1 time ---- 3323233 pairs aligned 0 times concordantly or discordantly; of the: 6646466 mates make up the pairs; of the: 2061887 (31.02%) aligned 0 times 216206 (3.25%) aligned exactly 1 timeyeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/

216206 (3.25%) aligned exactly 1 time

4368373 (65.72%) aligned >1 times

95.69% overall alignment rate

#回貼W251的雙端測序結果

perl: warning: Setting locale failed.

perl: warning: Plea check that your locale ttings:

LANGUAGE = "en_US:en",

LC_ALL = (unt),

LC_PAPER = "zh_-8",

LC_ADDRESS = "zh_-8",

LC_MONETARY = "zh_-8",

LC_NUMERIC = "zh_-8",

LC_TELEPHONE = "zh_-8",

LC_IDENTIFICATION = "zh_-8",

LC_MEASUREMENT = "zh_-8",

LC_TIME = "zh_-8",

LC_NAME = "zh_-8",

LANG = "en_-8"

are supported and installed on your system.

perl: warning: Falling back to the standard locale ("C").

25553075 reads; of the:

25553075 (100.00%) were paired; of the:

3705332 (14.50%) aligned concordantly 0 times

2003416 (7.84%) aligned concordantly exactly 1 time

19844327 (77.66%) aligned concordantly >1 times

----

3705332 pairs aligned concordantly 0 times; of the:

163553 (4.41%) aligned discordantly 1 time

----

3541779 pairs aligned 0 times concordantly or discordantly; of the:

7083558 mates make up the pairs; of the:

2021254 (28.53%) aligned 0 times

226959 (3.20%) aligned exactly 1 time

4835345 (68.26%) aligned >1 times

96.04% overall alignment rate

#回貼W252的雙端測序結果

perl: warning: Setting locale failed.

perl: warning: Plea check that your locale ttings:

LANGUAGE = "en_US:en",

LC_ALL = (unt),

LC_PAPER = "zh_-8",

LC_ADDRESS = "zh_-8",

LC_MONETARY = "zh_-8",

LC_NUMERIC = "zh_-8",

LC_TELEPHONE = "zh_-8",

LC_IDENTIFICATION = "zh_-8",

LC_MEASUREMENT = "zh_-8",

LC_TIME = "zh_-8",

LC_NAME = "zh_-8",

LANG = "en_-8"

are supported and installed on your system.

perl: warning: Falling back to the standard locale ("C").

24577100 reads; of the:

24577100 (100.00%) were paired; of the:

3173490 (12.91%) aligned concordantly 0 times

1898984 (7.73%) aligned concordantly exactly 1 time

19504626 (79.36%) aligned concordantly >1 times

----

3173490 pairs aligned concordantly 0 times; of the:

112017 (3.53%) aligned discordantly 1 time

----

3061473 pairs aligned 0 times concordantly or discordantly; of the:

6122946 mates make up the pairs; of the:

2060673 (33.65%) aligned 0 times

226885 (3.71%) aligned exactly 1 time

3835388 (62.64%) aligned >1 times

95.81% overall alignment rate

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bowtie2 -x -1 /home/yeyt/biodata/NH160034/NH160034/cleandata/asmbly/

這個過程比較消耗時間，我們于此同時做個簡單質量控制報告

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ $TRINITY_HOME/util/ >

#輸出到文件

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ cat

################################

## Counts of transcripts, etc.

################################

Total trinity 'genes': 110851

Total trinity transcripts: 220498

Percent GC: 42.98

########################################

Stats bad on ALL transcript contigs:

########################################

Contig N10: 4369

Contig N20: 3291

Contig N30: 2640

Contig N40: 2183

Contig N50: 1802

Median contig length: 542

Average contig: 1008.13

Total asmbled bas: 222289920

#####################################################

## Stats bad on ONLY LONGEST ISOFORM per 'GENE':

#####################################################

Contig N10: 3997

Contig N20: 2867

Contig N30: 2195

Contig N40: 1663

Contig N50: 1157

Median contig length: 364

Average contig: 686.86

Total asmbled bas: 76139520

解釋一下上面的結果。

首先做一個概括 拼接得到多少個基因，得到多少個轉錄本

然后平均的GC含量是多少

接下來做一個兩個工作

一個是基于所有轉錄本的contig統計

一個是基于所有基因的統計

N50代表的是

接下來我們將把得到的sam結果轉化成bam結果并進行排序以提供后期的分析文件m結果并進行排序以提供后期的分析文件

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ ls *sam | grep '25' |xargs -I [] echo 'samtools view -bS [] | samtools sort -o [].

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ cat

samtools view -bS | samtools sort -o

samtools view -bS | samtools sort -o

samtools view -bS R251sam | samtools sort -o

samtools view -bS | samtools sort -o

samtools view -bS | samtools sort -o

samtools view -bS | samtools sort -o

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bash

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ bash [bam_sort_core] merging from

[bam_sort_core] merging from

[bam_sort_core] merging from

[bam_sort_core] merging from

[bam_sort_core] merging from

[bam_sort_core] merging from

yeyt@ubuntu:~/biodata/NH160034/NH160034/cleandata/asmbly/trinity_out_dir$ ll | sort -nk 7

total 234179528

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:07

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:08

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:19 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:24 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 01:26 .jellyfish_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 .iworm_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 03:32 ed

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 partitioned_

-rw-rw-r-- 1 yeyt yeyt 0 Sep 14 10:39 recursive_

-rw-rw-r-- 1 yeyt yeyt 9 Sep 14 01:08 _count

-rw-rw-r-- 1 yeyt yeyt 10 Sep 14 01:59 _count

-rw-rw-r-- 1 yeyt yeyt 2757 Sep 14 10:31

-rw-rw-r-- 1 yeyt yeyt 22843 Sep 14 01:26

-rw-rw-r-- 1 yeyt yeyt 13366802 Sep 14 10:39 partitioned_

-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 14 10:39 recursive_

-rw-rw-r-- 1 yeyt yeyt 493022300 Sep 14 03:27

-rw-rw-r-- 1 yeyt yeyt Sep 14 01:08

-rw-rw-r-- 1 yeyt yeyt 26501596675 Sep 14 01:24

-rw-rw-r-- 1 yeyt yeyt 49568938526 Sep 14 06:38 scaffolding_

drwxrwxr-x 2 yeyt yeyt 4096 Sep 14 10:33 chrysalis/

drwxrwxr-x 3 yeyt yeyt 4096 Sep 14 01:03 insilico_read_normalization/

drwxrwxr-x 4 yeyt yeyt 4096 Sep 14 10:39 read_partitions/

-rw-rw-r-- 1 yeyt yeyt 0 Sep 15 20:51 align_

-rw-rw-r-- 1 yeyt yeyt 62 Sep 15 20:51

-rw-rw-r-- 1 yeyt yeyt 651 Sep 15 07:03

-rw-rw-r-- 1 yeyt yeyt 10213332 Sep 15 07:03 _trans_map

-rw-rw-r-- 1 yeyt yeyt 47753878 Sep 15 07:03 recursive_ted

-rw-rw-r-- 1 yeyt yeyt 244740565 Sep 15 07:03

drwxrwxr-x 3 yeyt yeyt 4096 Sep 15 18:42 ../

-rw-rw-r-- 1 yeyt yeyt 821 Sep 23 15:17

-rw-rw-r-- 1 yeyt yeyt 1984490 Sep 23 13:50 2

-rw-rw-r-- 1 yeyt yeyt 55572480 Sep 23 13:50 2

-rw-rw-r-- 1 yeyt yeyt 55572488 Sep 23 14:03 2

-rw-rw-r-- 1 yeyt yeyt 55572488 Sep 23 14:16 2

-rw-rw-r-- 1 yeyt yeyt 103828770 Sep 23 14:03 2

-rw-rw-r-- 1 yeyt yeyt 103828770 Sep 23 14:16 2

-rw-rw-r-- 1 yeyt yeyt 400 Sep 24 13:22

-rw-rw-r-- 1 yeyt yeyt 3049959975 Sep 24 15:37

-rw-rw-r-- 1 yeyt yeyt 3181086895 Sep 24 16:44

-rw-rw-r-- 1 yeyt yeyt 3192193677 Sep 24 15:06

-rw-rw-r-- 1 yeyt yeyt 3206939510 Sep 24 14:33

-rw-rw-r-- 1 yeyt yeyt 3267705730 Sep 24 16:11

-rw-rw-r-- 1 yeyt yeyt 3655386513 Sep 24 14:01

-rw-rw-r-- 1 yeyt yeyt 2 Sep 24 01:49

-rw-rw-r-- 1 yeyt yeyt 21235142607 Sep 24 02:03

-rw-rw-r-- 1 yeyt yeyt 21293400430 Sep 24 02:07 R251sam

-rw-rw-r-- 1 yeyt yeyt 21346715631 Sep 24 02:15

-rw-rw-r-- 1 yeyt yeyt 22197735984 Sep 24 02:29

-rw-rw-r-- 1 yeyt yeyt 24496840308 Sep 24 03:04

這樣我們就得到了6個sort后的bam文件

采用以下工具

bam_

clipping_

inner_

read_

read_