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Illumina data QC & b asic NGS tools
Содержание
- 1. Illumina data QC & b asic NGS tools
- 2. From the very beginning...AACCCGTACGTTTTGCAAACGACCGT...
- 3. From the very beginningSequencing...AACCCGTACGTTTTGCAAACGACCGT...AACCCGTACGTCGTACGTTTTGAACGACCGGTTTTGCAAACGGTACGTTTTGCA
- 4. From the very beginningSequencingCoverage...AACCCGTACGTTTTGCAAACGACCGT...AACCCGTACGTCGTACGTTTTGAACGACCGGTTTTGCAAACGGTACGTTTTGCA3x2x
- 5. From the very beginningSequencingCoverageErrorsMismatches...AACCCGTACGTTTTGCAAACGACCGT...AACCCGTTCGTCGTACGTTTTCAACGACCGGTTTTGCAAACGGTACGTTTTGCA
- 6. From the very beginningSequencingCoverageErrorsMismatchesIndels...AACCCGTACGTTTTGCAAACGACCGT...AACCCGTTCGTCGTACGTTTTTCAACGACCGGTTTTGCAAACGGTA_GTTTTGCA
- 7. Early daysSanger sequencingLong reads (~900 bp)Low coverage (< 10x)Extreme costHuman genome project3 Gbp3 billion USD10 years
- 8. NGSShorter reads (25-400bp)High coverage (50-1000x)Huge amount of data Low costMore applicationsRequired completely new algorithms
- 9. NGS technologies
- 10. Illumina sequencinghttp://www.youtube.com/watch?v=77r5p8IBwJk
- 11. IonTorrent sequencinghttps://www.youtube.com/watch?v=WYBzbxIfuKs
- 12. Paired readsAACCCGTACGTTTTGCAAACGACCGTAACCAAATTGGAACCCGTACGT........TAACCAAATTGGinsert sizePaired-end (< 1 kbp)Mate-pairs (1 - 20 kbp)
- 13. Insert size distributionInsert size# of reads
- 14. FASTA/FASTQFASTA>EAS20_8_6_1_9_1972/1ACCACCATTACCACCACCATCACCATTACCACAGGTAACGGTGCGGGCTGACGC>EAS20_8_6_1_163_1521/1GCAGAAAACGTTCTGCATTTGCCACTGATGTACCGCCGAACTTCAACACTCGCAFASTQ@EAS20_8_6_1_1477_92/1ACCGTTACCTGTGGTAATGGTGATGGTGGTGGTAATGGTGGTGCTAATGCGTTT+EAS20_8_6_1_1477_92/1HHGHFHHHHHHHHHGFFHHHBG?GGC8DD9GF??=FFBCGBAF>FGCFHGHGGGPhred qualityQ = [ - 10 log10 p / (1 - p) ]
- 15. seqtk utilitySubsampling sampleConverting between interleaved/paired files mergepe, seq -1/-2fastq->fasta seq -AQuality trimmingShifting the qualityModifying namesetc...
- 16. Quality Control
- 17. FastQCEasy and lightweight quality control for sequencing dataDoes not require reference genome
- 18. Per base sequence quality
- 19. Per base sequence quality
- 20. Per sequence GC content
- 21. Per sequence GC content
- 22. Per sequence GC content
- 23. Per base sequence content
- 24. Per base sequence content
- 25. FastQCfastqc -hmkdir fastqc … -o
- 26. Error correction
- 27. Per base sequence quality
- 28. TrimmomaticSE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36Remove leading
- 29. TrimmomaticScan the read with a 4-base wide
- 30. TrimmomaticPE OPTIONS ILLUMINACLIP: ILLUMINACLIP:TruSeq3-PE.fa
- 31. Adapter trimmingILLUMINACLIP:::: ILLUMINACLIP:NexteraPE-PE.fa:2:10:30
- 32. Thank you!
- 33. Скачать презентанцию
From the very beginning...AACCCGTACGTTTTGCAAACGACCGT...
Слайды и текст этой презентации
Слайд 3From the very beginning
Sequencing
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTACGT
CGTACGTTTTG
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA
Слайд 4From the very beginning
Sequencing
Coverage
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTACGT
CGTACGTTTTG
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA
3x
2x
Слайд 5From the very beginning
Sequencing
Coverage
Errors
Mismatches
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTTCGT
CGTACGTTTTC
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA
Слайд 6From the very beginning
Sequencing
Coverage
Errors
Mismatches
Indels
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTTCGT
CGTACGTTTTTC
AACGACCG
GTTTTGCAAACG
GTA_GTTTTGCA
Слайд 7Early days
Sanger sequencing
Long reads (~900 bp)
Low coverage (< 10x)
Extreme cost
Human
genome project
Слайд 8NGS
Shorter reads (25-400bp)
High coverage (50-1000x)
Huge amount of data
Low cost
More
applications
Required completely new algorithms
Слайд 12Paired reads
AACCCGTACGTTTTGCAAACGACCGTAACCAAATTGG
AACCCGTACGT........TAACCAAATTGG
insert size
Paired-end (< 1 kbp)
Mate-pairs (1 - 20 kbp)
Слайд 14FASTA/FASTQ
FASTA
>EAS20_8_6_1_9_1972/1
ACCACCATTACCACCACCATCACCATTACCACAGGTAACGGTGCGGGCTGACGC
>EAS20_8_6_1_163_1521/1
GCAGAAAACGTTCTGCATTTGCCACTGATGTACCGCCGAACTTCAACACTCGCA
FASTQ
@EAS20_8_6_1_1477_92/1
ACCGTTACCTGTGGTAATGGTGATGGTGGTGGTAATGGTGGTGCTAATGCGTTT
+EAS20_8_6_1_1477_92/1
HHGHFHHHHHHHHHGFFHHHBG?GGC8DD9GF??=FFBCGBAF>FGCFHGHGGG
Phred quality
Q = [ - 10 log10 p / (1
- p) ]
![FASTA/FASTQFASTA>EAS20_8_6_1_9_1972/1ACCACCATTACCACCACCATCACCATTACCACAGGTAACGGTGCGGGCTGACGC>EAS20_8_6_1_163_1521/1GCAGAAAACGTTCTGCATTTGCCACTGATGTACCGCCGAACTTCAACACTCGCAFASTQ@EAS20_8_6_1_1477_92/1ACCGTTACCTGTGGTAATGGTGATGGTGGTGGTAATGGTGGTGCTAATGCGTTT+EAS20_8_6_1_1477_92/1HHGHFHHHHHHHHHGFFHHHBG?GGC8DD9GF??=FFBCGBAF>FGCFHGHGGGPhred qualityQ = [ - 10 log10 p / (1 - p) ]](/img/thumbs/49b0795394e12f1a299824f27427c516-800x.jpg "Illumina data QC &
b asic NGS tools FASTA/FASTQFASTA>EAS20_8_6_1_9_1972/1ACCACCATTACCACCACCATCACCATTACCACAGGTAACGGTGCGGGCTGACGC>EAS20_8_6_1_163_1521/1GCAGAAAACGTTCTGCATTTGCCACTGATGTACCGCCGAACTTCAACACTCGCAFASTQ@EAS20_8_6_1_1477_92/1ACCGTTACCTGTGGTAATGGTGATGGTGGTGGTAATGGTGGTGCTAATGCGTTT+EAS20_8_6_1_1477_92/1HHGHFHHHHHHHHHGFFHHHBG?GGC8DD9GF??=FFBCGBAF>FGCFHGHGGGPhred qualityQ = [ - 10 log10 p / (1 - p) ]")
Слайд 15seqtk utility
Subsampling
sample
Converting between interleaved/paired files
mergepe, seq -1/-2
fastq->fasta
seq -A
Quality trimming
Shifting the
quality
Modifying names
etc...
Слайд 17FastQC
Easy and lightweight quality control for sequencing data
Does not require
reference genome
Слайд 28Trimmomatic
SE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Remove leading
low quality or N bases (below quality 3) (LEADING:3)
Remove trailing
low quality or N bases (below quality 3) (TRAILING:3)
Слайд 29Trimmomatic
Scan the read with a 4-base wide sliding window, cutting
when the average quality per base drops below 15 (SLIDINGWINDOW:4:15)
Drop
reads below the 36 bases long (MINLEN:36)
