Work Log 03/08

Transcript

1. Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine N G S Wo r k f l o w, Q u a l i t y C h e c k  Work Log – 03/08 From to
2. Current NGS workflow progress Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 2 *.bcl Demultiplexing Illumina CASAVA 1.8 FASTQ *.fastq FASTQ *.fastq FASTQ *.fastq Quality Check FastQC v0.10.1 HTML *.html Figs *.png Report *.txt QC & Trimming cutadapt FASTQ *.fastq
3. Demultiplexing - Barcode Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 3
4. Why the sequencer knows the beginning of a DNA (cDNA) insert? •  Only insert part of a read emits fluorescence •  But it does not know the end of an insert •  If insert is too short, the 3’ adapter may be read Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 4
5. Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 5 Quality Check Using FastQC v0.10.1
6. FastQC Report •  Basic Statistics •  Per base sequence quality •  Per sequence quality scores •  Per base sequence content •  Per base GC content •  Per sequence GC content •  Per base N content •  Sequence Length Distribution •  Sequence Duplication Levels •  Overrepresented sequences •  Kmer Content Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 6
7. Basic Statistics •  Encoding and its version Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 7
8. Per base sequence quality (good) •  X axis is not linear Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 8
9. Per base sequence quality (not good) •  need to trim quality Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 9
10. Per sequence quality scores (good) •  one peak at good score Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 10
11. Per sequence quality scores (not good) Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 11
12. Per base sequence content Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 12
13. Per base sequence content (not good) Bioinformatics and Biostatistics Core, NTU Center of Genomic Medicine 13