Lecture 5 - Computational analysis Flashcards
(41 cards)
It is now simple to measure the expression levels of thousands of genes
simultaneously
Methods such as RNA-seq allow for measurement of
transcriptome-wide expression levels without a reference genome
RNA-seq is useful for
high-throughput sequencing of RNA
RNA-seq allows for quantification of
gene expression and differential expression analyses
RNA-seq allows for characterization of
alternative splicing
de novo means
from the beginning
de novo transcriptome assembly allows for
quantification and exploration of boutique organisms (no genome sequence necessary)
RNA-seq steps
- Extraction of mRNA
- PCR amplification
- Sequencing (single or paired end)
Poly A selection is a method of
isolating Poly(A+) transcription usually using oligo-dT affinity
Ribodepletion depletes
ribosomal RNAs using sequence specific biotin-labeled probes
Reads
the sequenced portion of cDNA fragments
Coverage
read length, number of reads, or haploid genome length
Single-end
cDNA fragments are sequenced from only one end (1x100)
Paired-end
cDNA fragments are sequenced from both ends (2x100)
Strand-specific
You know whether the read originated from the + or - strand
Counts =
(Xi) the number of reads that align to a particular feature i (gene, isoform, miRNA, etc.)
Library size =
(N) number of reads sequenced
FPKM =
Fragments per kilobase of exon per million mapped reads
CPM =
Counts per million mapped reads
FDR =
False discovery rate (the rate of Type I errors - false positives)
FASTA files are
text files with sequences (amino acids or nucleotides)
FASTQ files are
text files containing header, sequence, and quality information
A SAM file is a
tab-delimited text file that contains sequence alignment information
BAM files are
the binary version (compressed and indexed version) of SAM files (they’re smaller)
