1. Introduction to NGS
Ana Conesa
Head of Genomics of Gene Expression Lab
Centro de Investigaciones Prínicpe Felipe
aconesa@cipf.es
http://bioinfo.cipf.es/aconesa
2. Next Generation Sequencing
NGS has brought high speed not only to genome sequencing
and personal medicine, but has also change the way we do
genome research:
Got a question on genome organization:
SEQUENCE IT!!!!
3. NGS technologies
Cost-effective
Fast
Ultra throughput
Cloning-free
Short reads
32. Some Figures
How much does it “cost” (computationally) to sequence a human transcriptome?
One human transcriptome: 100 Million reads
1 Solexa run ==8 lanes ==25 M reads/lane==2 x 4 G fastq/lane (PE)
32 G disk space
Mapping @ processor 12 cores, 48 GB RAM , 4TB disk 24 hours
SAM (Ascii) / BAM (Binary) output 36 G / 9 G
33. Applications of RNAseq
Qualitative:
Quantitative:
* Alternative splicing
* Differential expression
* Antisense expression
* Dynamic range of gene expression
* Extragenic expression
….
* Alternative 5’ and 3’ usage
* Detection of fusion transcripts
….
edgeR
Tophat/Cufflinks
DESeq
Scripture
baySeq
Alexa
NOISeq
34. Advantages of RNAseq?
RNAseq microarrays
* Non targeted transcript detection * Restricted to probes on array
* No need of reference genome * Needs genome knowledge
* Strand specificity * Normally, not strand specific
* Find novels splicing sites * Exon arrays difficult to use
* Larger dynamic range * Smaller dynamic range
* Detects expression and SNVs * Does not provide sequence info
* Detects rare transcripts * Rare transcripts difficult
…. ….
and…. are there any disadvantages?????
52. Conclusions
NGS is revolutionizing how
we do genome research
But it will also revolutionize
our lives….
If we manage to process
and analyze the data