Short read sequencing

Consensus calling and Structural variation

• Consensus generation and variant detection by Celera Assembler.
• Detecting Structural Variations, Brudno et al.

Read Mapping Software

• Short-Read_Sequence_Alignment programs

BFAST

• need to e-mail to author to get the code

BLAT

• BLAT—The BLAST-Like Alignment Tool, Genome Research 2002
• FAQ
• Can align any type of reads
• Can do nt:aa translation
• Command: blat

 blat -noHead -t=dna  -q=dna  -tileSize=10 -stepSize=3 Pa.1con    Pa.seq    Pa.blat

MAQ

• Maq Sourceforge
• Maq Poster from Sanger
• Illumina-Solexa/AB-SOLiD , not 454 or capillary reads
• Uses FASTQ format
• Command: maq map ...
• does ungapped alignment on unpaired reads

 SOLEXA
 maq.pl easyrun -d . ref.1con reads.fastq

 SOLID
 solid2fastq.pl reads_ shortname
 maq fastq2bfq shortname.fastq shortname.bfq
 maq fasta2csfa ref.fasta > ref.csfa
 maq fasta2bfa ref.csfa ref.csbfa
 maq fasta2bfa ref.fasta ref.bfa
 maq map -c aln.cs.map ref.csbfa shortname.bfq 2> aln.log
 maq csmap2nt aln.nt.map ref.bfa aln.cs.map
 maq assemble cns.cns ref.bfa aln.nt.map 2> cns.log

RMAP

• RMAP : designed for Illumina-Solexa
• Command: rmap

 rmap         -m 3 -w 33                            -c Pa.1con    Pa.seq -o Pa.rmap

SHRiMP

• Web site
• Commands: rmapper-cs , rmapper-ls, ...

SeqMap

• SeqMap developed at Stanford
• allows up to five mixed substitutions and inserted/deleted nucleotides in the mapping
• allows sequences to contain N’s, and to have unequal lengths

 ./seqmap
 Usage: seqmap <number of mismatches> <probe FASTA file name> <transcript FASTA file name> <output file name> [options]
 
 Parameters:
 <number of mismatches>                          maximum edit distance allowed
 <probe FASTA file name>                         probe/tag/read sequences
 <transcript FASTA file name>                    reference sequences
 <output file name>                              name of the output file
 ...

SHORE

• SHORE

SOAP

• Web site (China)
• Formatofoutput
• SOAP: short oligonucleotide alignment program, Bioinformatics Jan 2008
• Commands: soap, soap.contig, soap_dealign, soap.huge, soap.short
• can use qualities, do read trimming, use pair ends, RNA alignments

 soap         -v 5                                  -d Pa.1con -a Pa.seq -o Pa.soap

SOCS

• ABI color space

 socs socs.pref
 more socs.pref
 Req.fa
 Seq_F3.csfasta
 Seq_F3_QV.qual
 out_prefix
 2
 1000
 2
 false
 true
 0

SOLiD

• SOLID System Analysis Pipeline Tool (Corona Lite)

SSAHA

• Web site(Sanger)
• Focused on exact, nearly exact matches
• Does not find all the exact matches???
• Example: Solexa 33bp ~30% of reads are not found

ZOOM

• ZOOM

Genome Resequencing

• The complete genome of an individual by massively parallel DNA sequencing (J.Watson's genome) Nature April 2008
• J.Watson's genome (supplementary info)

• Whole-genome sequencing and variant discovery in C. elegans Nature Jan 2008

Links

• 1000 genomes

• Ben's web site 1
• Ben's web site 2

• Chip-Seq @ Wikipedia

• SRA
• SRA FTP

Data

Solexa

• Strep suis Solexa at Sanger 36bp, ~49X coverage
• Staphylococcus aureus strain MW2 (edena paper) 35bp, ~47X coverage
• Pseudomonas aeruginosa: 33bp, ~43X coverage
• Pseudomonas syringae: 32bp, ~31X coverage
• 1000 Genomes (June 14th 2008): 47bp

 Accession       #Runs   Instrument                      Center  Study                          [Individual]
 SRA000303       41      Solexa 1G Genome Analyzer       BI      1000Genomes Project Pilot 2     NA12878
 SRA000304       49      Solexa 1G Genome Analyzer       BI      1000Genomes Project Pilot 2     NA12891
 SRA000305       56      Solexa 1G Genome Analyzer       BI      1000Genomes Project Pilot 2     NA12892
 SRA000307       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA10851
 SRA000308       2       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA11993
 SRA000309       3       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA11995
 SRA000310       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12006
 SRA000311       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12044
 SRA000312       2       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12156
 SRA000313       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12414
 SRA000314       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12776
 SRA000315       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12828
 SRA000316       12      Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 2     NA12878
 SRA000317       8       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 2     NA12891
 SRA000318       14      Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 2     NA12892
 SRA000319       1       Solexa 1G Genome Analyzer       SC      1000Genomes Project Pilot 1     NA12004

June 14th 2008: Sept 19th 2008

 SRA001100       23      Illumina Genome Analyzer        BGI     1000Genomes Project Pilot 2     NA19240
 ...
 SRA002029       1       Illumina Genome Analyzer II     WUGSC   1000Genomes Project Pilot 2     NA19239

 /fs/szdata/Solexa/1000genomes

• Example SRR001113.seq :

 7,058,926 47 bp sequences
 2,402,398 contain at least 1 '.'

454

• 1000 Genomes

June 14th 2008

 Accession       #Runs   Instrument      Center  Study                           [Individual]
 SRA000302       121     454 GS FLX      BCM     1000Genomes Project Pilot 2     NA12878
 SRA001032       2       454 GS FLX      BCM     1000Genomes Project Pilot 2     NA12878
 SRA001036       1       454 GS FLX      BCM     1000Genomes Project Pilot 1     NA12812
 SRA001094       1       454 GS FLX      BCM     1000Genomes Project Pilot 2     NA12878

June 14th 2008: Sept 19th 2008

 SRA001037       2       454 GS FLX      BCM     1000Genomes Project Pilot 1     NA12812
 ...
 SRA001819       1       454 GS FLX      BCM     1000Genomes Project Pilot 2     NA12878

Refseq

• /fs/szdata/genomes/human_ncbi_build36/ NCBI build36.1 May 2006 (Current build is 36.3 March 2008)
• /fs/szdata/genomes/human_celera_2001_Orig/