有参转录组数据分析，样品和基因组比对效率只有零点几，这是为什么？

老师，您好，根据咱们转录组课程进行了实操，质控等正常，但是转录组数据比对到基因组这个步骤总是出现比对率只有零点几的情况，尝试了好几个转录组数据，从来没正常过。

水稻转录组测序数据显示如下，NCBI下载的单端数据：1 @SRR22419667.1 A00133:246:HNTYVDSXY:1:1101:30734:1454 length=300

2 TCCCGTTGAGGTTACACTCCAAATTGAATGATGAGGCAACGAATAACAAAGCAGCACTCAGAAATCCATGATGGTCCAGGCATCTACAAATGATATATCGCGCAACTCCATTTGAGTACAAATGGGTCCAAGCCCTGGCAACAGGTGTATCCTTAAGAG

3 +SRR22419667.1 A00133:246:HNTYVDSXY:1:1101:30734:1454 length=300

4 FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

5 @SRR22419667.2 A00133:246:HNTYVDSXY:1:1101:6822:1689 length=300

6 GAGCTCAGTAACAGCAGTCTCTGAGTCAGCATGCCATGTGAGTAAATCAACAAAAGGACAAGTACCAGGATCTAGCAAATTGGGTCAACTAGCTACACGGAGAAATTTAGAGACTGAAGTTTTGCAAAGTCAGCAAGTTAGCAGCCCAGCCAGTTCCAC

7 +SRR22419667.2 A00133:246:HNTYVDSXY:1:1101:6822:1689 length=300

8 FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF.

#水稻基因组的文件从ensemble plants上下载，构建索引的代码为

#基因组信息下载到服务器/home/us024/Imranrnaseq/my_rnaseq/ref

wget https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-61/fasta/oryza_sativa/dna/Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.gz

wget https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-61/gff3/oryza_sativa/Oryza_sativa.IRGSP-1.0.61.gff3.gz

gunzip -c Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.gz > Oryza_sativa.IRGSP-1.0.dna.toplevel.fa

gunzip -c Oryza_sativa.IRGSP-1.0.61.gff3.gz > Oryza_sativa.IRGSP-1.0.61.gff3

#修改gff3文件

sed 's#gene:##' Oryza_sativa.IRGSP-1.0.61.gff3|ee

sed 's#gene:##' Oryza_sativa.IRGSP-1.0.61.gff3|sed 's#transcript:##' |ee

#然后把修改后的另存为另一个名字，运行如下

sed 's#gene:##' Oryza_sativa.IRGSP-1.0.61.gff3|sed 's#transcript:##' > Oryza_sativa.IRGSP-1.0.61.gff31

#把源文件覆盖掉

mv Oryza_sativa.IRGSP-1.0.61.gff31 Oryza_sativa.IRGSP-1.0.61.gff3

#构建索引

sh $scriptdir/index.sh Oryza_sativa.IRGSP-1.0.dna.toplevel.fa Oryza_sativa.IRGSP-1.0.61.gff3

#构建索引后，gtf的部分显示，其它的生成的结果看着也没什么问题

1 1 RAP2022-09-01 transcript 2983 10815 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

2 1 RAP2022-09-01 exon 2983 3268 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

3 1 RAP2022-09-01 exon 3354 3616 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

4 1 RAP2022-09-01 exon 4357 4455 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

5 1 RAP2022-09-01 exon 5457 5560 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

6 1 RAP2022-09-01 exon 7136 7944 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

7 1 RAP2022-09-01 exon 8028 8150 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

8 1 RAP2022-09-01 exon 8232 8320 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

9 1 RAP2022-09-01 exon 8408 8608 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

10 1 RAP2022-09-01 exon 9210 9615 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

11 1 RAP2022-09-01 exon 10102 10187 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

12 1 RAP2022-09-01 exon 10274 10430 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

13 1 RAP2022-09-01 exon 10504 10815 . + . transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

14 1 RAP2022-09-01 CDS 3449 3616 . + 0 transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

15 1 RAP2022-09-01 CDS 4357 4455 . + 0 transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

16 1 RAP2022-09-01 CDS 5457 5560 . + 0 transcript_id "Os01t0100100-01"; gene_id "Os01g0100100";

#构建索引后，gff3的部分显示

69 #!genebuild-last-updated 2019-06

70 1 IRGSP-1.0 chromosome 1 43270923 . . . ID=chromosome:1;Alias=Chr1,AP014957.1,NC_029256.1

71 ###

72 1 RAP2022-09-01 gene 2983 10815 . + . ID=Os01g0100100;biotype=protein_coding;gene_id=Os01g0100100;logic_name=rapdb_genes

73 1 RAP2022-09-01 mRNA 2983 10815 . + . ID=Os01t0100100-01;Parent=Os01g0100100;biotype=protein_coding;tag=Ensembl_canonical;tra

74 1 RAP2022-09-01 exon 2983 3268 . + . Parent=Os01t0100100-01;Name=Os01t0100100-01-E1;constitutive=1;ensembl_end_phase=-1;ense

75 1 RAP2022-09-01 five_prime_UTR 2983 3268 . + . Parent=Os01t0100100-01

76 1 RAP2022-09-01 five_prime_UTR 3354 3448 . + . Parent=Os01t0100100-01

77 1 RAP2022-09-01 exon 3354 3616 . + . Parent=Os01t0100100-01;Name=Os01t0100100-01-E2;constitutive=1;ensembl_end_phase=0;ensem

78 1 RAP2022-09-01 CDS 3449 3616 . + 0 ID=CDS:Os01t0100100-01;Parent=Os01t0100100-01;protein_id=Os01t0100100-01

79 1 RAP2022-09-01 exon 4357 4455 . + . Parent=Os01t0100100-01;Name=Os01t0100100-01-E3;constitutive=1;ensembl_end_phase=0;ensem

80 1 RAP2022-09-01 CDS 4357 4455 . + 0 ID=CDS:Os01t0100100-01;Parent=Os01t0100100-01;protein_id=Os01t0100100-01

81 1 RAP2022-09-01 exon 5457 5560 . + . Parent=Os01t0100100-01;Name=Os01t0100100-01-E4;constitutive=1;ensembl_end_phase=2;ensem

#比对到基因组，先比对一个

hisat2 -p 3 --rg-id=400ppm_rep1 --rg SM:400ppm_rep1 --rg LB:400ppm_rep1 --rg PL:ILLUMINA -x $REF_INDEX --dta -U $workdir/2.data_qc/400ppm_rep1.clean.fq.gz -S 400ppm_rep1.sam 2> 400ppm_rep1.summary

#比对结果

24819002 reads; of these:

24819002 (100.00%) were unpaired; of these:

24775361 (99.82%) aligned 0 times

39911 (0.16%) aligned exactly 1 time

3730 (0.02%) aligned >1 times

0.18% overall alignment rate

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分类：转录组

有参转录组数据分析，样品和基因组比对效率只有零点几，这是为什么？

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