Jared - Revision history

24.224.85.54: /* Illumina Sequencing */

2011-01-23T01:09:25Z

‎Illumina Sequencing

24.224.85.54: /* 454 Sequencing */

2011-01-23T01:08:50Z

‎454 Sequencing

Jamimms: /* Read Length */

2011-01-20T20:51:39Z

‎Read Length

Jamimms: /* Read Length */

2011-01-20T20:50:53Z

‎Read Length

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-20T00:08:51Z

‎Single Molecule Real Time Sequencing

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-20T00:00:20Z

‎Single Molecule Real Time Sequencing

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-19T23:54:12Z

‎Single Molecule Real Time Sequencing

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-19T23:44:15Z

‎Single Molecule Real Time Sequencing

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-19T23:43:39Z

‎Single Molecule Real Time Sequencing

Jamimms: /* Single Molecule Real Time Sequencing */

2011-01-19T23:43:24Z

‎Single Molecule Real Time Sequencing

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 == Illumina Sequencing ==

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 == Read Length ==
-The issue with short reads (20-40 nt fragments sequenced at a time) is in assembly. We must use algorithms to find overlapping sections of fragments, then piece these fragments together.  There are many repetitive regions of the genome. Using only 20-40 nt fragments we may have a hard time finding overlapping regions and determining the correct linear chromosomal location of repetitive segments. While the error rate of sequencing is only 2 percent for the first 30 nucleotides at the head of reads using Illumina technology, the error rate quickly increases to 20 percent at the tails of reads at 50 nucleotides [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652199/]. The high error rate results from the incorporation of wrong bases by DNA polymerase (all four present at a time) with no error-fixing machinery found in a normal cell. Long reads are difficult because replicating DNA molecules in the clusters can get out of sync [http://www.genomesunzipped.org/2010/09/basics-second-generation-sequencing.php]. Paired end reads of circularized DNA of adapted kilobase fragments can be used to link repetitive segments to general location. The 454 Titanium FLX instrument can perform extra long reads of 400-600 bps to eliminate the need for paired ends [http://www.454.com/products-solutions/experimental-design-options/multi-span-paired-end-reads.asp]. Sequencing with short reads is generally cheaper and offers higher throughput, but, problems arise in ''de novo'' fragment assembly. Assembly from short read data is usually accomplished with the help of a reference genome [http://www.cseweb.ucsd.edu/~dbrinza/cv/pub/preprint.gr.079053.108.pdf], [http://www.cbcb.umd.edu/~salzberg/docs/AMOScmp-reprint.pdf]. The top two sets of ''de novo'' assembly algorithms are Beijing Genomics Institute's SOAPdenovo and the Broad Institute's ALLPATHS-LG [http://www.genomeweb.com//node/959135?hq_e=el&hq_m=904523&hq_l=1&hq_v=239c994d88].
+The issue with short reads (20-40 nt fragments sequenced at a time) is in assembly. We must use algorithms to find overlapping sections of fragments, then piece these fragments together.  There are many repetitive regions of the genome. Using only 20-40 nt fragments we may have a hard time finding overlapping regions and determining the correct linear chromosomal location of repetitive segments. While the error rate of sequencing is only 2 percent for the first 30 nucleotides at the head of reads using Illumina technology, the error rate quickly increases to 20 percent at the tails of reads at 50 nucleotides [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652199/]. The high error rate results from the incorporation of wrong bases by DNA polymerase (all four present at a time) with no error-fixing machinery found in a normal cell. Long reads are difficult because replicating, homogeneous DNA molecules in the clusters can get out of sync [http://www.genomesunzipped.org/2010/09/basics-second-generation-sequencing.php]. Paired end reads of circularized DNA of adapted kilobase fragments can be used to link repetitive segments to general location. The 454 Titanium FLX instrument can perform extra long reads of 400-600 bps to eliminate the need for paired ends [http://www.454.com/products-solutions/experimental-design-options/multi-span-paired-end-reads.asp]. Sequencing with short reads is generally cheaper and offers higher throughput, but, problems arise in ''de novo'' fragment assembly. Assembly from short read data is usually accomplished with the help of a reference genome [http://www.cseweb.ucsd.edu/~dbrinza/cv/pub/preprint.gr.079053.108.pdf], [http://www.cbcb.umd.edu/~salzberg/docs/AMOScmp-reprint.pdf]. The top two sets of ''de novo'' assembly algorithms are Beijing Genomics Institute's SOAPdenovo and the Broad Institute's ALLPATHS-LG [http://www.genomeweb.com//node/959135?hq_e=el&hq_m=904523&hq_l=1&hq_v=239c994d88].
 The good people from NCSU and the David H. Murdock Institute probably used the 454 FLX system to sequence the ''Vaccinium corymbosum'' genome ''de novo''. Longer reads produced by this system facilitate ''de novo'' assembly. They probably used an Illumina system to resequence and examine the quality of the assembly. This resequencing also increases coverage. The authors of "The genome of woodland strawberry (Fragaria vesca)" in ''Nature Genetics'' used a combination of Roche/454, Illumina/Solexa, and Life technologies/SOLiD to sequence and resequence [http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.740.html]. Curiously, Illumina advocates the use of the Genome Analyzer for ''de novo'' sequencing. Illumina points to over 100 bp reads achieved by some researchers. I question the accuracy of these reads. The company promotes the use of Velvet, a Bruijn graph-based assembly program [http://www.illumina.com/Documents/products/.../technote_denovo_assembly.pdf].

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 == Read Length ==
-The issue with short reads (20-40 nt fragments sequenced at a time) is in assembly. We must use algorithms to find overlapping sections of fragments, then piece these fragments together.  There are many repetitive regions of the genome. Using only 20-40 nt fragments we may have a hard time finding overlapping regions and determining the correct linear chromosomal location of repetitive segments. While the error rate of sequencing is only 2 percent for the first 30 nucleotides at the head of reads using Illumina technology, the error rate quickly increases to 20 percent at the tails of reads at 50 nucleotides [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652199/]. The high error rate results from the incorporation of wrong bases by DNA polymerase (all four present at a time) with no error-fixing machinery found in a normal cell. Long reads are difficult because DNA molecules in the clusters can get out of sync [http://www.genomesunzipped.org/2010/09/basics-second-generation-sequencing.php]. Paired end reads of circularized DNA of adapted kilobase fragments can be used to link repetitive segments to general location. The 454 Titanium FLX instrument can perform extra long reads of 400-600 bps to eliminate the need for paired ends [http://www.454.com/products-solutions/experimental-design-options/multi-span-paired-end-reads.asp]. Sequencing with short reads is generally cheaper and offers higher throughput, but, problems arise in ''de novo'' fragment assembly. Assembly from short read data is usually accomplished with the help of a reference genome [http://www.cseweb.ucsd.edu/~dbrinza/cv/pub/preprint.gr.079053.108.pdf], [http://www.cbcb.umd.edu/~salzberg/docs/AMOScmp-reprint.pdf]. The top two sets of ''de novo'' assembly algorithms are Beijing Genomics Institute's SOAPdenovo and the Broad Institute's ALLPATHS-LG [http://www.genomeweb.com//node/959135?hq_e=el&hq_m=904523&hq_l=1&hq_v=239c994d88].
+The issue with short reads (20-40 nt fragments sequenced at a time) is in assembly. We must use algorithms to find overlapping sections of fragments, then piece these fragments together.  There are many repetitive regions of the genome. Using only 20-40 nt fragments we may have a hard time finding overlapping regions and determining the correct linear chromosomal location of repetitive segments. While the error rate of sequencing is only 2 percent for the first 30 nucleotides at the head of reads using Illumina technology, the error rate quickly increases to 20 percent at the tails of reads at 50 nucleotides [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652199/]. The high error rate results from the incorporation of wrong bases by DNA polymerase (all four present at a time) with no error-fixing machinery found in a normal cell. Long reads are difficult because replicating DNA molecules in the clusters can get out of sync [http://www.genomesunzipped.org/2010/09/basics-second-generation-sequencing.php]. Paired end reads of circularized DNA of adapted kilobase fragments can be used to link repetitive segments to general location. The 454 Titanium FLX instrument can perform extra long reads of 400-600 bps to eliminate the need for paired ends [http://www.454.com/products-solutions/experimental-design-options/multi-span-paired-end-reads.asp]. Sequencing with short reads is generally cheaper and offers higher throughput, but, problems arise in ''de novo'' fragment assembly. Assembly from short read data is usually accomplished with the help of a reference genome [http://www.cseweb.ucsd.edu/~dbrinza/cv/pub/preprint.gr.079053.108.pdf], [http://www.cbcb.umd.edu/~salzberg/docs/AMOScmp-reprint.pdf]. The top two sets of ''de novo'' assembly algorithms are Beijing Genomics Institute's SOAPdenovo and the Broad Institute's ALLPATHS-LG [http://www.genomeweb.com//node/959135?hq_e=el&hq_m=904523&hq_l=1&hq_v=239c994d88].
 The good people from NCSU and the David H. Murdock Institute probably used the 454 FLX system to sequence the ''Vaccinium corymbosum'' genome ''de novo''. Longer reads produced by this system facilitate ''de novo'' assembly. They probably used an Illumina system to resequence and examine the quality of the assembly. This resequencing also increases coverage. The authors of "The genome of woodland strawberry (Fragaria vesca)" in ''Nature Genetics'' used a combination of Roche/454, Illumina/Solexa, and Life technologies/SOLiD to sequence and resequence [http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.740.html]. Curiously, Illumina advocates the use of the Genome Analyzer for ''de novo'' sequencing. Illumina points to over 100 bp reads achieved by some researchers. I question the accuracy of these reads. The company promotes the use of Velvet, a Bruijn graph-based assembly program [http://www.illumina.com/Documents/products/.../technote_denovo_assembly.pdf].

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 No clustering or washing is necessary, so long, cheap reads are possible. DNA libraries are incorporated into SMRTbell constructs of ligated circularized DNA bound to a single DNA polymerase molecule [http://www.nejm.org/doi/full/10.1056/NEJMoa1012928]. A laser is shined through the glass covering the zero-mode wavelength (ZMW) that excites only the 30nm bottom of the ZMW where the polymerase is actively incorporating fluorescently labeled nucleotides. Interference from non-incorporated nucleotides in the micropore is minimized. Accuracy of sequencing is still a major issue.
-A team at Harvard used PacBio's sequencing methods to sequence five strains of ''Vibrio colerae'' at 12X coverage in two days. That translates to approximately 368Mb/day throughput (of two strains). Unless my calculations are wrong, that throughput is on par with or worse than 454's throughput. Authors of the paper do not indicate cost of sequencing, assembly time, nor raw error rate. By matching PacBio raw reads to the ''Vibrio colerae'' reference genome, Dr. Elemento determined a raw error of 20 percent [http://oelemento.wordpress.com/2011/01/03/a-closer-look-at-the-first-pacbio-sequence-dataset/]. The average read length was more impressive: 954 bp. No paired end, no reference genome necessary for assembly [http://www.nejm.org/doi/full/10.1056/NEJMoa1012928]. With throughput of only 5.3Mb/30 minute run, the single molecule method is still toddling around. We will see how this technology progresses.
+A team at Harvard used PacBio's sequencing methods to sequence five strains of ''Vibrio colerae'' at varying coverages (in between 20X and 60X per strain) over two days. That translates to approximately 368Mb/day throughput (of two strains). That throughput is on par with or worse than 454's throughput. Authors of the paper do not indicate cost of sequencing, assembly time, nor raw error rate. By matching PacBio raw reads to the ''Vibrio colerae'' reference genome, Dr. Elemento determined a raw error of 20 percent [http://oelemento.wordpress.com/2011/01/03/a-closer-look-at-the-first-pacbio-sequence-dataset/]. The average read length was more impressive: 954 bp. No paired end, no reference genome necessary for assembly [http://www.nejm.org/doi/full/10.1056/NEJMoa1012928]. With throughput of only 5.3Mb/30 minute run, the single molecule method is still toddling around. We will see how this technology progresses.
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