一致性序列

ice-melt

Consensus sequence(最常见的碱基)

http://www.metagenomics.wiki/tools/samtools/consensus-sequence

https://samtools.github.io/bcftools/howtos/consensus-sequence.html

步骤

1)根据参比基因序列比对短读序列

# Create bowtie2 database
bowtie2-build REFERENCE.fasta REF_DB

# bowtie2 mapping
bowtie2 -x REF_DB -U SAMPLE.fastq --no-unal -S SAMPLE.sam

# samtools:  sort .sam file and convert to .bam file
samtools view -bS SAMPLE.sam | samtools sort - -o SAMPLE.bam

2)从.bam文件中获得consensus 序列

# 获得一致性序列，vcfutils.pl 是 bcftools程序的一个脚本
samtools mpileup -uf REFERENCE.fasta SAMPLE.bam | bcftools call -c | vcfutils.pl vcf2fq > SAMPLE_cns.fastq 

# 将 .fastq 转换为 .fasta ，设置碱基质量 lower than 20 to N
seqtk seq -aQ64 -q20 -n N SAMPLE_cns.fastq > SAMPLE_cns.fasta

 
# call variants
bcftools mpileup -Ou -f reference.fa alignments.bam | bcftools call -mv -Oz -o calls.vcf.gz
bcftools index calls.vcf.gz

# normalize indels
bcftools norm -f reference.fa calls.vcf.gz -Ob -o calls.norm.bcf

# filter adjacent indels within 5bp
bcftools filter --IndelGap 5 calls.norm.bcf -Ob -o calls.norm.flt-indels.bcf

ice-melt

@ice-melt

序列一致性指的是什么
https://zhuanlan.zhihu.com/p/55324279

ice-melt

@ice-melt

问题记录

使用 bowtie2 遇到的问题

issue1

bowtie2 -x /ceph_disk3/lx/temp/ref_idx/mngs_fmdv_genomic_db -1 /ceph_disk3/lx/temp/ref_idx/tt/r1.fastq -2 /ceph_disk3/lx/temp/ref_idx/tt/r2.fastq  --no-unal -S SAMPLE.sam 

Use of uninitialized value $bt2_args[0] in join or string at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 423.
Use of uninitialized value $bt2_args[1] in join or string at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 423.
Use of uninitialized value $_[0] in string eq at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 360.
Use of uninitialized value $_[1] in string eq at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 360.
Use of uninitialized value in exists at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 81.
Use of uninitialized value in exists at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 81.
Use of uninitialized value $bt2_args[0] in join or string at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 459.
Use of uninitialized value $bt2_args[1] in join or string at /home/bioinfo/miniconda2/envs/mpa/bin/bowtie2 line 459.
Saw ASCII character 10 but expected 33-based Phred qual.
terminate called after throwing an instance of 'int'
Aborted (core dumped)
(ERR): bowtie2-align exited with value 134

issue2

bowtie2 -S SAMPLE.sam -x /ceph_disk3/lx/temp/ref_idx/mngs_fmdv_genomic_db -U /ceph_disk3/lx/temp/ref_idx/tt/r1.fastq

Saw ASCII character 10 but expected 33-based Phred qual.
terminate called after throwing an instance of 'int'
Aborted (core dumped)
(ERR): bowtie2-align exited with value 134

问题是抽取的序列输出成fasta格式了，这里比对需要fastq格式

ice-melt

@ice-melt

针对issue1，该问题也有可能由
1） --no-unal 参数引起（去掉该参数运行正常）
2）质量评分有问题，可考虑 --phred64参数

anneng

Difference Between Conserved and Consensus Sequence
https://www.differencebetween.com/difference-between-conserved-and-consensus-sequence/

anneng

https://teaching.ncl.ac.uk/bms/wiki/index.php/Consensus_sequence

https://en.wikipedia.org/wiki/Promoter_(genetics)

anneng

Genetics:A Conceptual Approach