NGS data format and General Quality Control

1. NGS data format and General Quality Control

2. Data format “Flowchart”

3. Fastq file • Used to record raw reads coming off the sequencers • Each record contains four lines • Parameters were usually set by the sequencer, such as read length

4. Fastq file

5. Line 1 begins with a '@' character and is followed by a sequence identifier and an optional description (like a FASTA title line). • Line 2 is the raw sequence letters. The read length is the length of the string. • Line 3 begins with a '+' character and is optionally followed by the same sequence identifier (and any description) again. • Line 4 encodes the quality values for the sequence in Line 2, and must contain the same number of symbols as letters in the sequence. http://en.wikipedia.org/wiki/FASTQ_format

6. General quality control of raw reads • Using FASTQC • A tool that implements some general rules • 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

7. Quality scores

8. Perbase “N” percentage

9. Sample FASTQC reports Good quality : http://www.bioinformatics.babraham.ac.uk/projects/fastqc/good_sequence_short_fastqc/fastqc_report.html Bad quality: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/bad_sequence_fastqc/fastqc_report.html

10. Data format “Flowchart”

11. SAM/BAM • SAM stands for Sequence Alignment Map • BAM is the binary form of SAM • Used for mapped/aligned reads • Generated by NGS mapper/aligners

12. SAM

13. BAM