Post- sequencing quality evalua2on. or what to do when you get your reads from the sequencer

Transcription

1 Post- sequencing quality evalua2on or what to do when you get your reads from the sequencer

2 The fastq file contains informa2on about sequence and quality Read Iden(fier Sequence Quality

3 Sources of Library Read Quality Problems Sequencer problems Read quality Library problems GC content Library complexity Adaptor/primer contamina2on Ribosomal RNA

4 Evalua2ng Quality

5 FastQC Report (html) basics

6 Assessing Sequencing Quality

7 FastQC evaluates sequencer related quality in 3 different ways Per base sequence quality Average quality for each base pair Per 2le sequence quality Average spa2al quality on flow cell Per sequence quality score Average quality per read

8 FastQC evaluates sequencer related quality in 3 different ways Per base sequence quality Average quality for each base pair Per 2le sequence quality Average spa2al quality on flow cell Per sequence quality score Average quality per read

9 Per Base Sequence Quality

10 Mid- read drop in quality can effect mapping efficiency

11 FastQC evaluates sequencer related quality in 3 different ways Per base sequence quality Average quality for each base pair Per 2le sequence quality Average spa2al quality on flow cell Per sequence quality score Average quality per read

12 Per 2le sequence quality

13 Small loss of 2le quality

14 Large loss of flow cell quality

15 FastQC evaluates sequencer related quality in 3 different ways Per base sequence quality Average quality for each base pair Per 2le sequence quality Average spa2al quality on flow cell Per sequence quality score Average quality per read

16 Average quality per read

17 Drop in quality for a por2on of reads

18 Assessing Library Quality

19 Nucleo2de content of the reads Per base sequence content % nucleo2de representa2on at each bp Per sequence GC content Distribu2on of % GC content per read Per base N content % uncalled assigned nucleo2des (N) per posi2on Sequence length distribu2on

20 Nucleo2de content of the reads Per base sequence content % nucleo2de representa2on at each bp Per sequence GC content Distribu2on of % GC content per read Per base N content % uncalled assigned nucleo2des (N) per posi2on

21 Per base sequence content

22 Random hexamer bias

23 Adaptor/adaptor product

24 Nucleo2de content of the reads Per base sequence content % nucleo2de representa2on at each bp Per sequence GC content Distribu2on of % GC content per read Per base N content % uncalled assigned nucleo2des (N) per posi2on Sequence length distribu2on

25 GC content per read

26 Adaptor/Adaptor product

27 Biologically overrepresented sequence

28 Nucleo2de content of the reads Per base sequence content % nucleo2de representa2on at each bp Per sequence GC content Distribu2on of % GC content per read Per base N content % uncalled assigned nucleo2des (N) per posi2on Sequence length distribu2on

29 Per base N content

30 Nucleo2de content of the reads Per base sequence content % nucleo2de representa2on at each bp Per sequence GC content Distribu2on of % GC content per read Per base N content % uncalled assigned nucleo2des (N) per posi2on Sequence length distribu2on

31 Sequence length distribu2on

32 Length post- trimming

33 Library Content Sequence duplica2on levels Total sequences vs. De- duplicated sequence Overrepresented sequences Large polymer sequences Adaptor content % adapter per nucleo2de Kmer Content Overrepresented 5- mers

34 Library Content Sequence duplica2on levels Total sequences vs. Deduplicated sequence Overrepresented sequences Large polymer sequences Adaptor content % adapter per nucleo2de Kmer Content Overrepresented 5- mers

35 Sequence duplica2on levels

36 Low complexity Adapter/Adaptor reads

37 Biological sequence duplica2on

38 Library Content Sequence duplica2on levels Total sequences vs. Deduplicated sequence Overrepresented sequences Large polymer sequences Adaptor content % adapter per nucleo2de Kmer Content Overrepresented 5- mers

39 Overrepresented Sequences Ribosomal RNA

40 Library Content Sequence duplica2on levels Total sequences vs. Deduplicated sequence Overrepresented sequences Large polymer sequences Adaptor content % adapter per nucleo2de Kmer Content Overrepresented 5- mers

41 Adaptor Content

42 Too- small library fragments

43 Library Content Sequence duplica2on levels Total sequences vs. Deduplicated sequence Overrepresented sequences Large polymer sequences Adaptor content % adapter per nucleo2de Kmer Content Overrepresented 5- mers

44 K- mer (5- mer) content

45 Don t worry be happy!! Just because your library doesn t look perfect doesn t mean it is BAD. Trim reads Low quality or adapter reads Remove duplicates ONLY IF NECESSARY Mapping takes into account base quality Get more coverage

46 Trimming reads You can trim reads to remove adapters and low quality sequence We will cover how to do this in another video Here is a preview of how the library can change

47 Per base sequence content- before trimming

48 Per base sequence content- aber trimming

49 Per sequence GC content- Before trimming

50 Per sequence GC content- aber trimming

51 Overrepresented sequences Before trimming Aber trimming