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開始行:
[[Pythonバイオ]] [[Pythonバイオ/ツール]]~
&counter(); &lastmod();~
*リボソームRNA ribosomal RNA の除去 [#ze584e24]
-2019-07-23 ribosomal RNA
--[[RNA-seqにおけるrRNAの扱い - 備忘録 a record of inner life:http://amphipod.hatenablog.com/entry/2016/12/01/190843]]
--[[RNA-seq データからの rRNA除去 | Tips for NGS Data Analysis:http://catway.jp/bioinformatics/RNA-Seq/rrna.html]]
--[[RNA-Seq 〜研究に合わせたアプリケーションの選び方〜 (illumina):
https://jp.illumina.com/content/dam/illumina-marketing/apac/japan/documents/pdf/2015_techsupport_session7.pdf]]
--[[シーケンス講習会
RNA-seq library調製法の特徴と選び方（理研）:http://www.clst.riken.jp/files/5914/3338/5219/4_How_to_prepare_RNAseq_library_and_sequence_data.pdf]]
--[[rRNAのコンタミを除く SortMeRNA - macでインフォマティクス:
http://kazumaxneo.hatenablog.com/entry/2018/01/21/125838]]
--[[Bonsai bioinformatics - sortmerna
https://bioinfo.lifl.fr/sortmerna/sortmerna.php]]
--[[SortMeRNA-user-manual-v2.1.pdf:https://bioinfo.lifl.fr/RNA/sortmerna/code/SortMeRNA-user-manual-v2.1.pdf]]
対象は 16S, 23S, 5S
* 九大からのデータ [#h3c95016]
■ 岸本先生, 東邦大
SE51
E. coli
これまでの2ランの合計
file #リード数 ＃トリム済 #rrn_map #rrn_unmap %rrn_unmap 5M SEのnon-rRNAを得るために必要なSE数 5M SEのnon-rRNAを得るために不足しているSE数 5M SEのnon-rRNAを得るために必要なSE数に対する超過量
181012_10B_S7 17,054,354 17,048,531 15,372,799 1,675,732 9.83 50,886,281 33,831,927
181012_1p2-1_S11 14,637,538 14,632,750 11,399,254 3,233,496 22.09 22,634,229 7,996,691
181012_1p2-2_S13 17,575,234 17,568,939 13,697,563 3,871,376 22.03 22,698,950 5,123,716
181012_2p5-1_S12 11,690,525 11,687,060 8,966,637 2,720,423 23.27 21,486,594 9,796,069
181012_2p6-1_S14 16,662,621 16,657,233 12,576,242 4,080,991 24.49 20,414,920 3,752,299
181012_45a_plus_S2 18,990,853 18,985,343 14,055,624 4,929,719 25.96 19,261,598 270,745
181012_45b_plus_S3 15,055,824 15,052,797 11,717,495 3,335,302 22.15 22,570,406 7,514,582
181012_45c_plus_S4 21,339,929 21,336,596 16,477,436 4,859,160 22.77 21,958,455 618,526
181013_45A_minus_S18 16,532,044 16,524,826 6,155,460 10,369,366 62.72 7,971,579 *** 8,560,465
181019_43B_S1 14,923,538 14,920,375 10,969,417 3,950,958 26.47 18,885,974 3,962,436
181019_45A_plus_S5 16,869,266 16,865,671 12,115,849 4,749,822 28.16 17,757,788 888,522
181019_45L_S6 12,808,719 12,805,744 8,250,496 4,555,248 35.56 14,059,299 1,250,580
181019_45a10D_plus_S9 16,210,966 16,205,968 13,420,227 2,785,741 17.18 29,096,327 12,885,361
181019_45aIII6c_plus_S8 15,696,596 15,690,006 12,927,019 2,762,987 17.60 28,405,121 12,708,525
181019_45d7B_plus_S10 13,268,830 13,264,315 11,154,294 2,110,021 15.90 31,442,412 18,173,582
181026_10D_minus_S21 20,353,513 20,344,812 10,544,708 9,800,104 48.15 10,384,335 *** 9,969,178
181026_2-10B_minus_S20 20,487,318 20,476,914 9,177,453 11,299,461 55.15 9,065,617 *** 11,421,701
181026_45a_minus_S15 17,516,312 17,510,720 9,034,910 8,475,810 48.39 10,333,120 *** 7,183,192
181026_45b_minus_S16 15,943,050 15,937,770 7,184,469 8,753,301 54.90 9,106,879 *** 6,836,171
181026_45c_minus_S17 14,383,563 14,376,676 5,640,314 8,736,362 60.74 8,232,010 *** 6,151,553
181103_45alll6c_minus_S22 21,788,554 21,781,657 10,052,049 11,729,608 53.83 9,287,844 *** 12,500,710
181103_45d7B_minus_S23 22,149,677 22,140,700 16,750,927 5,389,773 24.33 20,547,876 *** 1,601,801
181103_Anc_S19 17,975,953 17,964,999 13,759,979 4,205,020 23.39 21,374,397 3,398,444
PwOw_minus_S25 18,853,776 18,849,002 8,300,103 10,548,899 55.95 8,936,371 *** 9,917,405
PwOw_plus_S24 19,269,944 19,263,287 14,659,892 4,603,395 23.89 20,930,144 1,660,200
123,832,204
16S, 23S, 5S
* sortmeRNAをやってみる 2019-07-23 [#a4350acb]
バイナリをダウンロード、/usr/local/bin/sortmernaからリンク
データベースは？ ディレクトリ ~/src/sortmerna-2.1b/rRNA_databasesを確認
silva-arc-16s-id95.fasta arcaea 古細菌
silva-arc-23s-id98.fasta
silva-bac-16s-id90.fasta bacterium 細菌
silva-bac-23s-id98.fasta
silva-euk-18s-id95.fasta eukaryota 真核生物
silva-euk-28s-id98.fasta
rfam-5s-database-id98.fasta
rfam-5.8s-database-id98.fasta
じゃあ、silva-bac-16s-id90.fasta と silva-bac-23s-id98.fasta と rfam-5s-database-id98.fastaでいいのか。
indexdbを作る
cd ~/src/sortmerna-2.1b
indexdb_rna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:\
./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:\
./rRNA_databases/silva-arc-16s-id95.fasta,./index/silva-arc-16s-db:\
./rRNA_databases/silva-arc-23s-id98.fasta,./index/silva-arc-23s-db:\
./rRNA_databases/silva-euk-18s-id95.fasta,./index/silva-euk-18s-db:\
./rRNA_databases/silva-euk-28s-id98.fasta,./index/silva-euk-28s:\
./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:\
./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db
時間がかかるが、１回だけ行っておけばいい（らしい）。indexディレクトリ下にファイルができた。
これを使って１つだけ処理。たとえば、
~/src/KishimotoRNA2/181012_10B_S7_R1_001.trim.fastq
を試してみる。
sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:\
./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:\
./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:\
./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db \
--reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA \
--other 181012_10B_S7_R1_001.trim.non_rRNA --log -v
画面
Program: SortMeRNA version 2.1b, 03/03/2016
Copyright: 2012-16 Bonsai Bioinformatics Research Group:
LIFL, University Lille 1, CNRS UMR 8022, INRIA Nord-Europe
2014-16 Knight Lab:
Department of Pediatrics, UCSD, La Jolla,
Disclaimer: SortMeRNA comes with ABSOLUTELY NO WARRANTY; without even the
implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.
See the GNU Lesser General Public License for more details.
Contact: Evguenia Kopylova, jenya.kopylov@gmail.com
Laurent Noé, laurent.noe@lifl.fr
Hélène Touzet, helene.touzet@lifl.fr
Computing read file statistics ... done [23.20 sec]
size of reads file: 2906733904 bytes
partial section(s) to be executed: 3 of size 1073741824 bytes
Parameters summary:
Number of seeds = 2
Edges = 4 (as integer)
SW match = 2
SW mismatch = -3
SW gap open penalty = 5
SW gap extend penalty = 2
SW ambiguous nucleotide = -3
SQ tags are not output
Number of threads = 1
Begin mmap reads section # 1:
Time to mmap reads and set up pointers [1.37 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602725
Gumbel K = 0.329559
Minimal SW score based on E-value = 59
Loading index part 1/1 ... done [1.34 sec]
Begin index search ... done [719.52 sec]
Freeing index ... done [0.24 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602436
Gumbel K = 0.335011
Minimal SW score based on E-value = 58
Loading index part 1/1 ... done [1.00 sec]
Begin index search ... done [1471.22 sec]
Freeing index ... done [0.16 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.616694
Gumbel K = 0.342032
Minimal SW score based on E-value = 56
Loading index part 1/1 ... done [0.60 sec]
Begin index search ... done [143.74 sec]
Freeing index ... done [0.08 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.617555
Gumbel K = 0.343861
Minimal SW score based on E-value = 54
Loading index part 1/1 ... done [0.22 sec]
Begin index search ... done [111.06 sec]
Freeing index ... done [0.03 sec]
Total number of reads mapped (incl. all reads file sections searched): 5800992
Writing aligned FASTA/FASTQ ... done [18.38 sec]
Writing not-aligned FASTA/FASTQ ... done [1.82 sec]
Begin mmap reads section # 2:
Time to mmap reads and set up pointers [1.53 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Loading index part 1/1 ... done [1.44 sec]
Begin index search ... done [690.19 sec]
Freeing index ... done [0.27 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Loading index part 1/1 ... done [0.93 sec]
Begin index search ... done [1429.56 sec]
Freeing index ... done [0.14 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Loading index part 1/1 ... done [0.58 sec]
Begin index search ... done [140.79 sec]
Freeing index ... done [0.07 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Loading index part 1/1 ... done [0.21 sec]
Begin index search ... done [111.21 sec]
Freeing index ... done [0.02 sec]
Total number of reads mapped (incl. all reads file sections searched): 11563780
Writing aligned FASTA/FASTQ ... done [19.83 sec]
Writing not-aligned FASTA/FASTQ ... done [2.13 sec]
Begin mmap reads section # 3:
Time to mmap reads and set up pointers [1.07 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Loading index part 1/1 ... done [1.42 sec]
Begin index search ... done [523.39 sec]
Freeing index ... done [0.26 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Loading index part 1/1 ... done [0.93 sec]
Begin index search ... done [1039.06 sec]
Freeing index ... done [0.18 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Loading index part 1/1 ... done [0.60 sec]
Begin index search ... done [100.63 sec]
Freeing index ... done [0.07 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Loading index part 1/1 ... done [0.22 sec]
Begin index search ... done [80.05 sec]
Freeing index ... done [0.02 sec]
Total number of reads mapped (incl. all reads file sections searched): 15633981
Writing aligned FASTA/FASTQ ... done [13.92 sec]
Writing not-aligned FASTA/FASTQ ... done [1.40 sec]
ログファイル 181012_10B_S7_R1_001.rRNA.log
Tue Jul 23 16:59:31 2019
Command: sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db --reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA --other 181012_10B_S7_R1_001.trim.non_rRNA.fastq fastq -v
Process pid = 70967
Parameters summary:
Index: ./index/silva-bac-16s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602725
Gumbel K = 0.329559
Minimal SW score based on E-value = 59
Index: ./index/silva-bac-23s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602436
Gumbel K = 0.335011
Minimal SW score based on E-value = 58
Index: ./index/rfam-5s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.616694
Gumbel K = 0.342032
Minimal SW score based on E-value = 56
Index: ./index/rfam-5.8s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.617555
Gumbel K = 0.343861
Minimal SW score based on E-value = 54
Number of seeds = 2
Edges = 4 (as integer)
SW match = 2
SW mismatch = -3
SW gap open penalty = 5
SW gap extend penalty = 2
SW ambiguous nucleotide = -3
SQ tags are not output
Number of threads = 1
Reads file = 181012_10B_S7_R1_001.trim.fastq
Results:
Total reads = 17049704
Total reads passing E-value threshold = 15633981 (91.70%)
Total reads failing E-value threshold = 1415723 (8.30%)
Minimum read length = 36
Maximum read length = 51
Mean read length = 50
By database:
./rRNA_databases/silva-bac-16s-id90.fasta 20.37%
./rRNA_databases/silva-bac-23s-id98.fasta 71.31%
./rRNA_databases/rfam-5s-database-id98.fasta 0.02%
./rRNA_databases/rfam-5.8s-database-id98.fasta 0.00%
Tue Jul 23 18:50:05 2019
トリム前
wc -l 181012_10B_S7_R1_001.fastq
68217416 181012_10B_S7_R1_001.fastq 4で割ると 17,054,354
トリム後
wc -l 181012_10B_S7_R1_001.trim.fastq
68198816 181012_10B_S7_R1_001.trim.fastq 4で割ると 17,049,704
作られたfastqファイルの長さ
wc -l 181012_10B_S7_R1_001.rRNA.fastq で測定
62,535,924 181012_10B_S7_R1_001.rRNA.fastq 4で割ると 15,633,981
5,662,892 181012_10B_S7_R1_001.trim.non_rRNA.fastq 4で割ると 1,415,723
足すと 17,049,704
九大からの情報
file #リード数 ＃トリム済 #rrn_map #rrn_unmap %rrn_unmap
181012_10B_S7 17,054,354 17,048,531 15,372,799 1,675,732 9.83
分離後得られたr-RNAのsamファイル 181012_10B_S7_R1_001.rRNA.sam の先頭を取り出して覗いてみる。
head -500 181012_10B_S7_R1_001.rRNA.sam > 181012_10B_S7_R1_001.rRNA-500.sam
@HD VN:1.0 SO:unsorted
@PG ID:sortmerna VN:1.0 CL:sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db --reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA --other 181012_10B_S7_R1_001.trim.non_rRNA.fastq fastq -v
C00122:247:HLWJLBCX2:1:1101:10535:2128 0 FU8Pinit 117 255 1S47M3S * 0 0 TATTGCTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGG DDDDDIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIIIHIIII AS:i:79 NM:i:3
C00122:247:HLWJLBCX2:1:1101:12824:2040 0 FU8Pinit 121 255 49M2S * 0 0 CTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTAC DDDDDIIIIGIIHIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:78 NM:i:4
C00122:247:HLWJLBCX2:1:1101:10750:2503 0 FU8Pinit 123 255 47M4S * 0 0 AGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGA DDDDDHHIIHIIHIIIIIIIIIIIIIIIIIGIHIIHIIHIIIIGIIIIIID AS:i:74 NM:i:4
C00122:247:HLWJLBCX2:1:1101:8130:2894 0 Unc13453 1436 255 5S3M1D43M * 0 0 CCTACGGTTACCTTGTTTCGACTTCACCCCAGTCATGAATCACAAAGTGGT DDDDDIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:82 NM:i:2
C00122:247:HLWJLBCX2:1:1101:19062:2832 0 FU8Pinit 70 255 36M1D15M * 0 0 GTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGACCCACGATTAC DDDDDIIIIIIIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIHIIIIHIIII AS:i:62 NM:i:8
C00122:247:HLWJLBCX2:1:1101:6236:3120 0 FU8Pinit 134 255 4S36M11S * 0 0 CCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTT DDDDDHIIIIIIGHIIIIIIHIIHIHIIIIIIIIIIHGHHIHIIIIIIIII AS:i:62 NM:i:2
C00122:247:HLWJLBCX2:1:1101:3119:3328 0 FU8Pinit 81 255 25M1D25M1S * 0 0 CCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCG DDDDDIHIIIIHIIIIIIIIIIIIIIIHIIIIIIIHIIIIIIHIIIIIIIH AS:i:75 NM:i:5
C00122:247:HLWJLBCX2:1:1101:6916:3312 0 FU8Pinit 75 255 1S31M1D19M * 0 0 ACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGC DDDDDIIIIIGHHDHIHIIIIIIIIIIHIGIIIIIIIIIIIIIHIHIHIII AS:i:70 NM:i:6
C00122:247:HLWJLBCX2:1:1101:14490:3742 0 Unc13453 1436 255 6S3M1D42M * 0 0 CCCTACGGTTACCTTGTTCCGACTTCACCCCAGTCATGAATCACAAAGTGG DDDDDHHGHHHIIIHHIIGHIIIHHGIHIHIFHHHHHH?FHHEHHGHHIII AS:i:80 NM:i:2
C00122:247:HLWJLBCX2:1:1101:10644:3794 0 FU8Pinit 75 255 31M1D20M * 0 0 CAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCG DDDDDHIIIIIHIIIIIHHIIIIIIIIIHIIIIIIIIIIIIIIIHIIIHII AS:i:72 NM:i:6
C00122:247:HLWJLBCX2:1:1101:12145:3862 0 FU8Pinit 51 255 49M2S * 0 0 CCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGG DDDDDIIIIIIIIIHHIIIIIIIIIIIIHIIIIIIIIIIIIIIIHIIIIII AS:i:73 NM:i:5
C00122:247:HLWJLBCX2:1:1101:14381:3795 0 FU8Pinit 124 255 46M5S * 0 0 GCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGAC DDDDDIIIIGHIIIHEHHIGHIIIGIIIIIIIIHHHIIIIIIDIIIIIIII AS:i:72 NM:i:4
C00122:247:HLWJLBCX2:1:1101:20932:3891 0 Unc13453 1436 255 5S3M1D43M * 0 0 CCTACGGTTACCTTGTTCCGACTTCACCCCAGTCATGAATCACAAAGTGGT DDDDDHHIIIIIHHIHIIHIIIIIIIIIHHIHFHHHHIIHGIHIIIIIIII AS:i:82 NM:i:2
C00122:247:HLWJLBCX2:1:1101:20311:4173 0 FU8Pinit 59 255 1S47M1D3M * 0 0 GTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTG <D@DBCHIDHI<CEHIIHEGHIIHIIIGHHIDHH?EHHIGHIFHII?FHGH AS:i:75 NM:i:5
C00122:247:HLWJLBCX2:1:1101:9967:4272 0 I6CCervi 5 255 1S43M7S * 0 0 CCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCATGATC DDDDDIIHIHIIHHIIIIHIIIIIIIIIIIIIHIIIIIIIIHIIIIIIIII AS:i:86 NM:i:0
C00122:247:HLWJLBCX2:1:1101:5980:4705 0 FU8Pinit 52 255 48M3S * 0 0 CATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGC DDDDDIIIIHHIIIIIIIIIIIIIIIIHHIIIIIIIIIIIIIIIIIIIIII AS:i:76 NM:i:4
C00122:247:HLWJLBCX2:1:1101:7670:4720 0 FU8Pinit 123 255 47M4S * 0 0 AGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGA DDDDDIIIIIHIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIII AS:i:74 NM:i:4
C00122:247:HLWJLBCX2:1:1101:14848:4727 0 Unc13453 1436 255 1S3M1D47M * 0 0 CGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGC DDDDDIIIIIIIIIIIIIIIHIIIH<EH<GHHIGIIIIIIIIIIHIIIIII AS:i:90 NM:i:2
non-rRNAのfastqファイルを、マッピングして、どれだけ二重マップがあるか出してみる。
nohup hisat2 -p 16 --dta-cufflinks -x AP012030.fna -U 181012_10B_S7_R1_001.trim.non_rRNA.fastq -S 181012_10B_S7_R1_001.trim.non_rRNA.sam > 181012_10B_S7_R1_001.trim.non_rRNA.hisat2.log &
logファイルでは
1415723 reads; of these:
1415723 (100.00%) were unpaired; of these:
30855 (2.18%) aligned 0 times
1366651 (96.53%) aligned exactly 1 time
18217 (1.29%) aligned >1 times
97.82% overall alignment rate
ということで、>1 timesが1.29%で、OKになったみたいである。なおこの処理はあっという間に終わった。
終了行:
[[Pythonバイオ]] [[Pythonバイオ/ツール]]~
&counter(); &lastmod();~
*リボソームRNA ribosomal RNA の除去 [#ze584e24]
-2019-07-23 ribosomal RNA
--[[RNA-seqにおけるrRNAの扱い - 備忘録 a record of inner life:http://amphipod.hatenablog.com/entry/2016/12/01/190843]]
--[[RNA-seq データからの rRNA除去 | Tips for NGS Data Analysis:http://catway.jp/bioinformatics/RNA-Seq/rrna.html]]
--[[RNA-Seq 〜研究に合わせたアプリケーションの選び方〜 (illumina):
https://jp.illumina.com/content/dam/illumina-marketing/apac/japan/documents/pdf/2015_techsupport_session7.pdf]]
--[[シーケンス講習会
RNA-seq library調製法の特徴と選び方（理研）:http://www.clst.riken.jp/files/5914/3338/5219/4_How_to_prepare_RNAseq_library_and_sequence_data.pdf]]
--[[rRNAのコンタミを除く SortMeRNA - macでインフォマティクス:
http://kazumaxneo.hatenablog.com/entry/2018/01/21/125838]]
--[[Bonsai bioinformatics - sortmerna
https://bioinfo.lifl.fr/sortmerna/sortmerna.php]]
--[[SortMeRNA-user-manual-v2.1.pdf:https://bioinfo.lifl.fr/RNA/sortmerna/code/SortMeRNA-user-manual-v2.1.pdf]]
対象は 16S, 23S, 5S
* 九大からのデータ [#h3c95016]
■ 岸本先生, 東邦大
SE51
E. coli
これまでの2ランの合計
file #リード数 ＃トリム済 #rrn_map #rrn_unmap %rrn_unmap 5M SEのnon-rRNAを得るために必要なSE数 5M SEのnon-rRNAを得るために不足しているSE数 5M SEのnon-rRNAを得るために必要なSE数に対する超過量
181012_10B_S7 17,054,354 17,048,531 15,372,799 1,675,732 9.83 50,886,281 33,831,927
181012_1p2-1_S11 14,637,538 14,632,750 11,399,254 3,233,496 22.09 22,634,229 7,996,691
181012_1p2-2_S13 17,575,234 17,568,939 13,697,563 3,871,376 22.03 22,698,950 5,123,716
181012_2p5-1_S12 11,690,525 11,687,060 8,966,637 2,720,423 23.27 21,486,594 9,796,069
181012_2p6-1_S14 16,662,621 16,657,233 12,576,242 4,080,991 24.49 20,414,920 3,752,299
181012_45a_plus_S2 18,990,853 18,985,343 14,055,624 4,929,719 25.96 19,261,598 270,745
181012_45b_plus_S3 15,055,824 15,052,797 11,717,495 3,335,302 22.15 22,570,406 7,514,582
181012_45c_plus_S4 21,339,929 21,336,596 16,477,436 4,859,160 22.77 21,958,455 618,526
181013_45A_minus_S18 16,532,044 16,524,826 6,155,460 10,369,366 62.72 7,971,579 *** 8,560,465
181019_43B_S1 14,923,538 14,920,375 10,969,417 3,950,958 26.47 18,885,974 3,962,436
181019_45A_plus_S5 16,869,266 16,865,671 12,115,849 4,749,822 28.16 17,757,788 888,522
181019_45L_S6 12,808,719 12,805,744 8,250,496 4,555,248 35.56 14,059,299 1,250,580
181019_45a10D_plus_S9 16,210,966 16,205,968 13,420,227 2,785,741 17.18 29,096,327 12,885,361
181019_45aIII6c_plus_S8 15,696,596 15,690,006 12,927,019 2,762,987 17.60 28,405,121 12,708,525
181019_45d7B_plus_S10 13,268,830 13,264,315 11,154,294 2,110,021 15.90 31,442,412 18,173,582
181026_10D_minus_S21 20,353,513 20,344,812 10,544,708 9,800,104 48.15 10,384,335 *** 9,969,178
181026_2-10B_minus_S20 20,487,318 20,476,914 9,177,453 11,299,461 55.15 9,065,617 *** 11,421,701
181026_45a_minus_S15 17,516,312 17,510,720 9,034,910 8,475,810 48.39 10,333,120 *** 7,183,192
181026_45b_minus_S16 15,943,050 15,937,770 7,184,469 8,753,301 54.90 9,106,879 *** 6,836,171
181026_45c_minus_S17 14,383,563 14,376,676 5,640,314 8,736,362 60.74 8,232,010 *** 6,151,553
181103_45alll6c_minus_S22 21,788,554 21,781,657 10,052,049 11,729,608 53.83 9,287,844 *** 12,500,710
181103_45d7B_minus_S23 22,149,677 22,140,700 16,750,927 5,389,773 24.33 20,547,876 *** 1,601,801
181103_Anc_S19 17,975,953 17,964,999 13,759,979 4,205,020 23.39 21,374,397 3,398,444
PwOw_minus_S25 18,853,776 18,849,002 8,300,103 10,548,899 55.95 8,936,371 *** 9,917,405
PwOw_plus_S24 19,269,944 19,263,287 14,659,892 4,603,395 23.89 20,930,144 1,660,200
123,832,204
16S, 23S, 5S
* sortmeRNAをやってみる 2019-07-23 [#a4350acb]
バイナリをダウンロード、/usr/local/bin/sortmernaからリンク
データベースは？ ディレクトリ ~/src/sortmerna-2.1b/rRNA_databasesを確認
silva-arc-16s-id95.fasta arcaea 古細菌
silva-arc-23s-id98.fasta
silva-bac-16s-id90.fasta bacterium 細菌
silva-bac-23s-id98.fasta
silva-euk-18s-id95.fasta eukaryota 真核生物
silva-euk-28s-id98.fasta
rfam-5s-database-id98.fasta
rfam-5.8s-database-id98.fasta
じゃあ、silva-bac-16s-id90.fasta と silva-bac-23s-id98.fasta と rfam-5s-database-id98.fastaでいいのか。
indexdbを作る
cd ~/src/sortmerna-2.1b
indexdb_rna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:\
./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:\
./rRNA_databases/silva-arc-16s-id95.fasta,./index/silva-arc-16s-db:\
./rRNA_databases/silva-arc-23s-id98.fasta,./index/silva-arc-23s-db:\
./rRNA_databases/silva-euk-18s-id95.fasta,./index/silva-euk-18s-db:\
./rRNA_databases/silva-euk-28s-id98.fasta,./index/silva-euk-28s:\
./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:\
./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db
時間がかかるが、１回だけ行っておけばいい（らしい）。indexディレクトリ下にファイルができた。
これを使って１つだけ処理。たとえば、
~/src/KishimotoRNA2/181012_10B_S7_R1_001.trim.fastq
を試してみる。
sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:\
./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:\
./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:\
./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db \
--reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA \
--other 181012_10B_S7_R1_001.trim.non_rRNA --log -v
画面
Program: SortMeRNA version 2.1b, 03/03/2016
Copyright: 2012-16 Bonsai Bioinformatics Research Group:
LIFL, University Lille 1, CNRS UMR 8022, INRIA Nord-Europe
2014-16 Knight Lab:
Department of Pediatrics, UCSD, La Jolla,
Disclaimer: SortMeRNA comes with ABSOLUTELY NO WARRANTY; without even the
implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.
See the GNU Lesser General Public License for more details.
Contact: Evguenia Kopylova, jenya.kopylov@gmail.com
Laurent Noé, laurent.noe@lifl.fr
Hélène Touzet, helene.touzet@lifl.fr
Computing read file statistics ... done [23.20 sec]
size of reads file: 2906733904 bytes
partial section(s) to be executed: 3 of size 1073741824 bytes
Parameters summary:
Number of seeds = 2
Edges = 4 (as integer)
SW match = 2
SW mismatch = -3
SW gap open penalty = 5
SW gap extend penalty = 2
SW ambiguous nucleotide = -3
SQ tags are not output
Number of threads = 1
Begin mmap reads section # 1:
Time to mmap reads and set up pointers [1.37 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602725
Gumbel K = 0.329559
Minimal SW score based on E-value = 59
Loading index part 1/1 ... done [1.34 sec]
Begin index search ... done [719.52 sec]
Freeing index ... done [0.24 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602436
Gumbel K = 0.335011
Minimal SW score based on E-value = 58
Loading index part 1/1 ... done [1.00 sec]
Begin index search ... done [1471.22 sec]
Freeing index ... done [0.16 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.616694
Gumbel K = 0.342032
Minimal SW score based on E-value = 56
Loading index part 1/1 ... done [0.60 sec]
Begin index search ... done [143.74 sec]
Freeing index ... done [0.08 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.617555
Gumbel K = 0.343861
Minimal SW score based on E-value = 54
Loading index part 1/1 ... done [0.22 sec]
Begin index search ... done [111.06 sec]
Freeing index ... done [0.03 sec]
Total number of reads mapped (incl. all reads file sections searched): 5800992
Writing aligned FASTA/FASTQ ... done [18.38 sec]
Writing not-aligned FASTA/FASTQ ... done [1.82 sec]
Begin mmap reads section # 2:
Time to mmap reads and set up pointers [1.53 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Loading index part 1/1 ... done [1.44 sec]
Begin index search ... done [690.19 sec]
Freeing index ... done [0.27 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Loading index part 1/1 ... done [0.93 sec]
Begin index search ... done [1429.56 sec]
Freeing index ... done [0.14 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Loading index part 1/1 ... done [0.58 sec]
Begin index search ... done [140.79 sec]
Freeing index ... done [0.07 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Loading index part 1/1 ... done [0.21 sec]
Begin index search ... done [111.21 sec]
Freeing index ... done [0.02 sec]
Total number of reads mapped (incl. all reads file sections searched): 11563780
Writing aligned FASTA/FASTQ ... done [19.83 sec]
Writing not-aligned FASTA/FASTQ ... done [2.13 sec]
Begin mmap reads section # 3:
Time to mmap reads and set up pointers [1.07 sec]
Begin analysis of: ./rRNA_databases/silva-bac-16s-id90.fasta
Loading index part 1/1 ... done [1.42 sec]
Begin index search ... done [523.39 sec]
Freeing index ... done [0.26 sec]
Begin analysis of: ./rRNA_databases/silva-bac-23s-id98.fasta
Loading index part 1/1 ... done [0.93 sec]
Begin index search ... done [1039.06 sec]
Freeing index ... done [0.18 sec]
Begin analysis of: ./rRNA_databases/rfam-5s-database-id98.fasta
Loading index part 1/1 ... done [0.60 sec]
Begin index search ... done [100.63 sec]
Freeing index ... done [0.07 sec]
Begin analysis of: ./rRNA_databases/rfam-5.8s-database-id98.fasta
Loading index part 1/1 ... done [0.22 sec]
Begin index search ... done [80.05 sec]
Freeing index ... done [0.02 sec]
Total number of reads mapped (incl. all reads file sections searched): 15633981
Writing aligned FASTA/FASTQ ... done [13.92 sec]
Writing not-aligned FASTA/FASTQ ... done [1.40 sec]
ログファイル 181012_10B_S7_R1_001.rRNA.log
Tue Jul 23 16:59:31 2019
Command: sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db --reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA --other 181012_10B_S7_R1_001.trim.non_rRNA.fastq fastq -v
Process pid = 70967
Parameters summary:
Index: ./index/silva-bac-16s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602725
Gumbel K = 0.329559
Minimal SW score based on E-value = 59
Index: ./index/silva-bac-23s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.602436
Gumbel K = 0.335011
Minimal SW score based on E-value = 58
Index: ./index/rfam-5s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.616694
Gumbel K = 0.342032
Minimal SW score based on E-value = 56
Index: ./index/rfam-5.8s-db
Seed length = 18
Pass 1 = 18, Pass 2 = 9, Pass 3 = 3
Gumbel lambda = 0.617555
Gumbel K = 0.343861
Minimal SW score based on E-value = 54
Number of seeds = 2
Edges = 4 (as integer)
SW match = 2
SW mismatch = -3
SW gap open penalty = 5
SW gap extend penalty = 2
SW ambiguous nucleotide = -3
SQ tags are not output
Number of threads = 1
Reads file = 181012_10B_S7_R1_001.trim.fastq
Results:
Total reads = 17049704
Total reads passing E-value threshold = 15633981 (91.70%)
Total reads failing E-value threshold = 1415723 (8.30%)
Minimum read length = 36
Maximum read length = 51
Mean read length = 50
By database:
./rRNA_databases/silva-bac-16s-id90.fasta 20.37%
./rRNA_databases/silva-bac-23s-id98.fasta 71.31%
./rRNA_databases/rfam-5s-database-id98.fasta 0.02%
./rRNA_databases/rfam-5.8s-database-id98.fasta 0.00%
Tue Jul 23 18:50:05 2019
トリム前
wc -l 181012_10B_S7_R1_001.fastq
68217416 181012_10B_S7_R1_001.fastq 4で割ると 17,054,354
トリム後
wc -l 181012_10B_S7_R1_001.trim.fastq
68198816 181012_10B_S7_R1_001.trim.fastq 4で割ると 17,049,704
作られたfastqファイルの長さ
wc -l 181012_10B_S7_R1_001.rRNA.fastq で測定
62,535,924 181012_10B_S7_R1_001.rRNA.fastq 4で割ると 15,633,981
5,662,892 181012_10B_S7_R1_001.trim.non_rRNA.fastq 4で割ると 1,415,723
足すと 17,049,704
九大からの情報
file #リード数 ＃トリム済 #rrn_map #rrn_unmap %rrn_unmap
181012_10B_S7 17,054,354 17,048,531 15,372,799 1,675,732 9.83
分離後得られたr-RNAのsamファイル 181012_10B_S7_R1_001.rRNA.sam の先頭を取り出して覗いてみる。
head -500 181012_10B_S7_R1_001.rRNA.sam > 181012_10B_S7_R1_001.rRNA-500.sam
@HD VN:1.0 SO:unsorted
@PG ID:sortmerna VN:1.0 CL:sortmerna --ref ./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db --reads 181012_10B_S7_R1_001.trim.fastq --sam --num_alignments 1 --fastx --aligned 181012_10B_S7_R1_001.rRNA --other 181012_10B_S7_R1_001.trim.non_rRNA.fastq fastq -v
C00122:247:HLWJLBCX2:1:1101:10535:2128 0 FU8Pinit 117 255 1S47M3S * 0 0 TATTGCTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGG DDDDDIIIIIIIIIIIIIIIIIIIIIIIIGIIIIIIIIIIIIIIIIHIIII AS:i:79 NM:i:3
C00122:247:HLWJLBCX2:1:1101:12824:2040 0 FU8Pinit 121 255 49M2S * 0 0 CTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTAC DDDDDIIIIGIIHIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:78 NM:i:4
C00122:247:HLWJLBCX2:1:1101:10750:2503 0 FU8Pinit 123 255 47M4S * 0 0 AGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGA DDDDDHHIIHIIHIIIIIIIIIIIIIIIIIGIHIIHIIHIIIIGIIIIIID AS:i:74 NM:i:4
C00122:247:HLWJLBCX2:1:1101:8130:2894 0 Unc13453 1436 255 5S3M1D43M * 0 0 CCTACGGTTACCTTGTTTCGACTTCACCCCAGTCATGAATCACAAAGTGGT DDDDDIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:82 NM:i:2
C00122:247:HLWJLBCX2:1:1101:19062:2832 0 FU8Pinit 70 255 36M1D15M * 0 0 GTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGACCCACGATTAC DDDDDIIIIIIIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIHIIIIHIIII AS:i:62 NM:i:8
C00122:247:HLWJLBCX2:1:1101:6236:3120 0 FU8Pinit 134 255 4S36M11S * 0 0 CCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTT DDDDDHIIIIIIGHIIIIIIHIIHIHIIIIIIIIIIHGHHIHIIIIIIIII AS:i:62 NM:i:2
C00122:247:HLWJLBCX2:1:1101:3119:3328 0 FU8Pinit 81 255 25M1D25M1S * 0 0 CCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCG DDDDDIHIIIIHIIIIIIIIIIIIIIIHIIIIIIIHIIIIIIHIIIIIIIH AS:i:75 NM:i:5
C00122:247:HLWJLBCX2:1:1101:6916:3312 0 FU8Pinit 75 255 1S31M1D19M * 0 0 ACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGC DDDDDIIIIIGHHDHIHIIIIIIIIIIHIGIIIIIIIIIIIIIHIHIHIII AS:i:70 NM:i:6
C00122:247:HLWJLBCX2:1:1101:14490:3742 0 Unc13453 1436 255 6S3M1D42M * 0 0 CCCTACGGTTACCTTGTTCCGACTTCACCCCAGTCATGAATCACAAAGTGG DDDDDHHGHHHIIIHHIIGHIIIHHGIHIHIFHHHHHH?FHHEHHGHHIII AS:i:80 NM:i:2
C00122:247:HLWJLBCX2:1:1101:10644:3794 0 FU8Pinit 75 255 31M1D20M * 0 0 CAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCG DDDDDHIIIIIHIIIIIHHIIIIIIIIIHIIIIIIIIIIIIIIIHIIIHII AS:i:72 NM:i:6
C00122:247:HLWJLBCX2:1:1101:12145:3862 0 FU8Pinit 51 255 49M2S * 0 0 CCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGG DDDDDIIIIIIIIIHHIIIIIIIIIIIIHIIIIIIIIIIIIIIIHIIIIII AS:i:73 NM:i:5
C00122:247:HLWJLBCX2:1:1101:14381:3795 0 FU8Pinit 124 255 46M5S * 0 0 GCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGAC DDDDDIIIIGHIIIHEHHIGHIIIGIIIIIIIIHHHIIIIIIDIIIIIIII AS:i:72 NM:i:4
C00122:247:HLWJLBCX2:1:1101:20932:3891 0 Unc13453 1436 255 5S3M1D43M * 0 0 CCTACGGTTACCTTGTTCCGACTTCACCCCAGTCATGAATCACAAAGTGGT DDDDDHHIIIIIHHIHIIHIIIIIIIIIHHIHFHHHHIIHGIHIIIIIIII AS:i:82 NM:i:2
C00122:247:HLWJLBCX2:1:1101:20311:4173 0 FU8Pinit 59 255 1S47M1D3M * 0 0 GTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTG <D@DBCHIDHI<CEHIIHEGHIIHIIIGHHIDHH?EHHIGHIFHII?FHGH AS:i:75 NM:i:5
C00122:247:HLWJLBCX2:1:1101:9967:4272 0 I6CCervi 5 255 1S43M7S * 0 0 CCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCATGATC DDDDDIIHIHIIHHIIIIHIIIIIIIIIIIIIHIIIIIIIIHIIIIIIIII AS:i:86 NM:i:0
C00122:247:HLWJLBCX2:1:1101:5980:4705 0 FU8Pinit 52 255 48M3S * 0 0 CATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGC DDDDDIIIIHHIIIIIIIIIIIIIIIIHHIIIIIIIIIIIIIIIIIIIIII AS:i:76 NM:i:4
C00122:247:HLWJLBCX2:1:1101:7670:4720 0 FU8Pinit 123 255 47M4S * 0 0 AGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGA DDDDDIIIIIHIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIII AS:i:74 NM:i:4
C00122:247:HLWJLBCX2:1:1101:14848:4727 0 Unc13453 1436 255 1S3M1D47M * 0 0 CGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGC DDDDDIIIIIIIIIIIIIIIHIIIH<EH<GHHIGIIIIIIIIIIHIIIIII AS:i:90 NM:i:2
non-rRNAのfastqファイルを、マッピングして、どれだけ二重マップがあるか出してみる。
nohup hisat2 -p 16 --dta-cufflinks -x AP012030.fna -U 181012_10B_S7_R1_001.trim.non_rRNA.fastq -S 181012_10B_S7_R1_001.trim.non_rRNA.sam > 181012_10B_S7_R1_001.trim.non_rRNA.hisat2.log &
logファイルでは
1415723 reads; of these:
1415723 (100.00%) were unpaired; of these:
30855 (2.18%) aligned 0 times
1366651 (96.53%) aligned exactly 1 time
18217 (1.29%) aligned >1 times
97.82% overall alignment rate
ということで、>1 timesが1.29%で、OKになったみたいである。なおこの処理はあっという間に終わった。
ページ名:
![[Toho University]](image/CLogo_Kihon_process.jpg "[Toho University]")
![[YYLAB]](image/yamanouc2.png "[YYLAB]")
