1. NGS data format and General Quality Control

2. Data format “Flowchart” Sequencer raw data FastqSAM/BAM

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).FASTA 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 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_s equence_fastqc/fastqc_report.html http://www.bioinformatics.babraham.ac.uk/projects/fastqc/bad_s equence_fastqc/fastqc_report.html

10. Data format “Flowchart” SequencerFastqSAM/BAM

11. 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