# 目录
1.Module 1 - Introduction to RNA sequencing
2.Module 2 - RNA-seq Alignment and Visualization
3.Module 3 - Expression and Differential Expression
4.Module 4 - Isoform Discovery and Alternative Expression
- Reference Guided Transcript Assembly
- de novo Transcript Assembly
- Transcript Assembly Merge
- Differential Splicing
- Splicing Visualization
5.Module 5 - De novo transcript reconstruction
6.Module 6 - Functional Annotation of Transcripts
# 2.5 Alignment QC
# 使用 samtools 和 FastQC 来评估比对结果
使用 samtools view 查看 SAM/BAM 比对文件的格式
samtools view -H UHR.bam | |
samtools view UHR.bam | head |
尝试过滤 BAM 文件以排除某些标记。这可以通过 samtools view -f -F 选项实现
-f INT required flag -F INT filtering flag
Try requiring that alignments are 'paired' and 'mapped in a proper pair' (=3). Also filter out alignments that are 'unmapped', the 'mate is unmapped', and 'not primary alignment' (=268)
samtools view -f 3 -F 268 UHR.bam | head |
现在要求只对 “PCR or optical duplicate” 进行比对。有多少 reads 符合这个标准？为什么？
samtools view -f 1024 UHR.bam | head |
使用 samtools flagstat 获取比对的基本情况。比对到上 reads 的百分比是多少？
samtools flagstat UHR.bam | |
1174953 + 0 in total (QC-passed reads + QC-failed reads) | |
24539 + 0 secondary | |
0 + 0 supplementary | |
0 + 0 duplicates | |
1173741 + 0 mapped (99.90% : N/A) | |
1150414 + 0 paired in sequencing | |
575207 + 0 read1 | |
575207 + 0 read2 | |
1143860 + 0 properly paired (99.43% : N/A) | |
1148598 + 0 with itself and mate mapped | |
604 + 0 singletons (0.05% : N/A) | |
6 + 0 with mate mapped to a different chr | |
6 + 0 with mate mapped to a different chr (mapQ>=5) | |
samtools flagstat HBR.bam | |
793963 + 0 in total (QC-passed reads + QC-failed reads) | |
7597 + 0 secondary | |
0 + 0 supplementary | |
0 + 0 duplicates | |
793356 + 0 mapped (99.92% : N/A) | |
786366 + 0 paired in sequencing | |
393183 + 0 read1 | |
393183 + 0 read2 | |
783124 + 0 properly paired (99.59% : N/A) | |
785496 + 0 with itself and mate mapped | |
263 + 0 singletons (0.03% : N/A) | |
0 + 0 with mate mapped to a different chr | |
0 + 0 with mate mapped to a different chr (mapQ>=5) |
SAM/BAM 格式的详细信息可以在这里找到:http://samtools.sourceforge.net/SAM1.pdf
# Using FastQC
你可以使用 FastQC 来执行基本的 BAM 文件的 QC (Pre-Alignment QC)。输出非常类似于在 fastq 文件上运行 FastQC。
