Illumina data QC & basic NGS tools презентация

Август 6, 2022

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Illumina data QC & basic NGS tools

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2. From the very beginning ...AACCCGTACGTTTTGCAAACGACCGT...
3. From the very beginning Sequencing ...AACCCGTACGTTTTGCAAACGACCGT... AACCCGTACGT CGTACGTTTTG AACGACCG GTTTTGCAAACG GTACGTTTTGCA
4. From the very beginning Sequencing Coverage ...AACCCGTACGTTTTGCAAACGACCGT... AACCCGTACGT CGTACGTTTTG AACGACCG GTTTTGCAAACG GTACGTTTTGCA 3x 2x
5. From the very beginning Sequencing Coverage Errors Mismatches ...AACCCGTACGTTTTGCAAACGACCGT... AACCCGTTCGT CGTACGTTTTC AACGACCG GTTTTGCAAACG GTACGTTTTGCA
6. From the very beginning Sequencing Coverage Errors Mismatches Indels ...AACCCGTACGTTTTGCAAACGACCGT... AACCCGTTCGT CGTACGTTTTTC AACGACCG GTTTTGCAAACG GTA_GTTTTGCA
7. Early days Sanger sequencing Long reads (~900 bp) Low coverage ( Extreme cost Human genome project
8. NGS Shorter reads (25-400bp) High coverage (50-1000x) Huge amount of data Low cost More applications Required
9. NGS technologies
10. Illumina sequencing http://www.youtube.com/watch?v=77r5p8IBwJk
11. IonTorrent sequencing https://www.youtube.com/watch?v=WYBzbxIfuKs
12. Paired reads AACCCGTACGTTTTGCAAACGACCGTAACCAAATTGG AACCCGTACGT........TAACCAAATTGG insert size Paired-end ( Mate-pairs (1 - 20 kbp)
13. Insert size distribution Insert size # of reads
14. FASTA/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 = [
15. seqtk utility Subsampling sample Converting between interleaved/paired files mergepe, seq -1/-2 fastq->fasta seq -A Quality trimming
16. Quality Control
17. FastQC Easy and lightweight quality control for sequencing data Does 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. FastQC fastqc -h mkdir fastqc … -o
26. Error correction
27. Per base sequence quality
28. Trimmomatic SE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 Remove leading low quality or N bases (below quality 3)
29. Trimmomatic Scan the read with a 4-base wide sliding window, cutting when the average quality per
30. Trimmomatic PE OPTIONS ILLUMINACLIP: ILLUMINACLIP:TruSeq3-PE.fa
31. Adapter trimming ILLUMINACLIP: : : threshold>: ILLUMINACLIP:NexteraPE-PE.fa:2:10:30
33. Скачать презентацию

Слайд 2

From the very beginning
...AACCCGTACGTTTTGCAAACGACCGT...

Слайд 3

From the very beginning
Sequencing
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTACGT
CGTACGTTTTG
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA

Слайд 4

From the very beginning
Sequencing
Coverage
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTACGT
CGTACGTTTTG
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA
3x
2x

Слайд 5

From the very beginning
Sequencing
Coverage
Errors
Mismatches
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTTCGT
CGTACGTTTTC
AACGACCG
GTTTTGCAAACG
GTACGTTTTGCA

Слайд 6

From the very beginning
Sequencing
Coverage
Errors
Mismatches
Indels
...AACCCGTACGTTTTGCAAACGACCGT...
AACCCGTTCGT
CGTACGTTTTTC
AACGACCG
GTTTTGCAAACG
GTA_GTTTTGCA

Слайд 7

Early days
Sanger sequencing
Long reads (~900 bp)
Low coverage (< 10x)
Extreme cost
Human genome

project
3 Gbp
3 billion USD
10 years

Слайд 8

NGS
Shorter reads (25-400bp)
High coverage (50-1000x)
Huge amount of data
Low cost
More applications
Required

completely new algorithms

Слайд 9

NGS technologies

Слайд 10

Illumina sequencing
http://www.youtube.com/watch?v=77r5p8IBwJk

Слайд 11

IonTorrent sequencing
https://www.youtube.com/watch?v=WYBzbxIfuKs

Слайд 12

Paired reads
AACCCGTACGTTTTGCAAACGACCGTAACCAAATTGG
AACCCGTACGT........TAACCAAATTGG
insert size
Paired-end (< 1 kbp)
Mate-pairs (1 - 20 kbp)

Слайд 13

Insert size distribution
Insert size
# of reads

Слайд 14

FASTA/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) ]

Слайд 15

seqtk 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 16

Quality Control

Слайд 17

FastQC
Easy and lightweight quality control for sequencing data
Does not require reference

genome

Слайд 18

Per base sequence quality

Слайд 19

Per base sequence quality

Слайд 20

Per sequence GC content

Слайд 21

Per sequence GC content

Слайд 22

Per sequence GC content

Слайд 23

Per base sequence content

Слайд 24

Per base sequence content

Слайд 25

FastQC
fastqc -h
mkdir
fastqc … -o

Слайд 26

Error correction

Слайд 27

Per base sequence quality

Слайд 28

Trimmomatic
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 29

Trimmomatic
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)

Слайд 30

Trimmomatic
PE

OPTIONS
ILLUMINACLIP:
ILLUMINACLIP:TruSeq3-PE.fa

Слайд 31

Adapter trimming
ILLUMINACLIP:::threshold>:
ILLUMINACLIP:NexteraPE-PE.fa:2:10:30