blobtools鉴定转录组污染来源

作者: 徒唤奈何_c5f0 | 来源:发表于2023-12-11 14:49 被阅读0次

全长转录本结构分析（下）
科研干货 | 一文了解RNA序列比对软件HISAT2
1.5构建支原体index并比对
【代谢组学】2.代谢物鉴定
DAP-seq
单细胞质控和标准化
动物转录因子注释和预测
动物转录因子注释和预测
10x 空间转录组最新文献集锦
转录组学习三（数据质控）

方法参考De Novo Transcriptome Assembly and Analysis of the Flat Oyster Pathogenic Protozoa Bonamia Ostreae这篇文章中对转录组内容的描述。

Transcriptome Taxonomic Assignment

Taxonomic assigment of the whole collection of the de novo-assembled transcripts was performed with two different tools: (i) with MEGAN version 6.20.18 (Huson et al., 2016) after a DIAMOND-blastx (version 2.0.6) interrogation of the mRNA transcripts with parameters: –sensitive –max-target-seqs 100 –e value 1e-3 -F 15 –range-culling against the RefSeq protein database, accessed in February 2021 (Buchfink et al., 2021); and (ii) with Blobtool2 implemented in BlobToolKit version 1.3.6 (Challis et al., 2020) after a BLASTn (version 2.9.0) interrogation of the mRNA transcripts against the nt database accessed in February 2021 with the following parameters: -max_hsps 1 -max_target_seqs 10 -evalue 1e-25 and a DIAMOND-blastx interrogation of the mRNA transcripts against the RefSeq protein database with parameters: –sensitive –max-target-seqs 1 –evalue 1e-25 (version 2.0.6, accessed in February 2021).

Taxonomic assignment of transcripts performed with BlobToolKit after a DIAMOND-BlastX interrogation of the RefSeq database and a BlastN interrogation of the nt database. A blob plot of length versus GC proportion for each transcript was created. Records are colored by phylum. Circles are sized in proportion to TPM on a square-root scale. Histograms show the distribution of TPM sum along each axis.

一本地构建diamond库

需要nr数据，prot.accession2taxid.gz，taxdump/nodes.dmp（可根据自己物种建库，需nr数据）

diamond makedb --in db_nr/nr_plants/plants.nr.fa.gz --db db_nr/db_nr_plants/nr_plants --taxonmap db_nr/prot.accession2taxid.gz --taxonnodes db_nr/taxdump/nodes.dmp
//总的来说，建库文件约为fa文件的两倍大

二比对

根据blobtools官网修改命令。

diamond blastx \
        --query assembly.fasta \
        --db /path/to/uniprot.db.with.taxids \
        --outfmt 6 qseqid staxids bitscore qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore \
        --sensitive \
        --max-target-seqs 1 \
        --evalue 1e-25 \
        --threads 16 \
        > diamond.out
//修改如下
diamond blastx --query rna/Trinity.fasta --db db_nr_plants/nr_plants1.dmnd        --outfmt 6 qseqid staxids bitscore qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore         --sensitive --max-target-seqs 1 --evalue 1e-25 --threads 16 -o diamond_blast_hits/plants_nr
Total time = 37934.5s
Reported 29673 pairwise alignments, 29673 HSPs.
29673 queries aligned.

三文件夹和添加数据

blobtools create \
    --fasta /path/to/assembly.fasta \
    --taxid 13804 \
    --taxdump ~/taxdump \
    AssemblyName
blobtools add \
    --hits blast.out \
    --hits diamond.out \
    --taxrule bestsumorder \
    --taxdump ~/taxdump \
    AssemblyName
blobtools add \
//实际代码
blobtools create --fasta rna/Trinity_HB.fasta blobtools_dir/Trinity_blob
blobtools add --hits diamond_blast_hits/plants_nr  --taxdump taxdump --taxrule bestsumorder blobtools_dir/Trinity_blob
blobtools host `pwd`
//运行后打开本地浏览器，可进行可视化绘图

问题解决

#blobtools安装时出现多重问题，最终重新安装wsl ubuntu22.04，conda新建python3.9的环境bkb，并且使用pip安装gcc
sudo apt-get update
sudo apt-get install build-essential
//安装blobtools2 ，重启conda环境后可使用
blobtools
BlobTools2 - assembly exploration, QC and filtering.
usage: blobtools [<command>] [<args>...] [-h|--help] [--version]

##使用diamond建库遇到问题，Accession exceeds supported length
查验后发现是prot.accession2taxid.gz的问题，且diamond版本太旧，使用conda重新安装diamond
conda create -n diamond1
conda activate diamond1
conda config --add channels conda-forge
conda install libgcc-ng=12 //gcc依赖版本太低，需要升级gcc
conda install -c bioconda diamond=2.1.8

gc-length_blob