CAGEscan mapping protocol: Difference between revisions
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(Be more verbose on each step of the CAGEscan pipeline (to be continued).) |
(Simplified sample splitting and linker removal.) |
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Input is 5′ and 3′ paired-end fastq files from the Illumina sequencers. |
Input is 5′ and 3′ paired-end fastq files from the Illumina sequencers. |
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=== 5′ === |
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| ⚫ | * The 6 first bases of the 3′ reads are trimmed because they derive from to the random part (N6) of the reverse-transcription primer, and therefore may not reflect the RNA sequences accurately, since the reverse-transcriptase tolerates mismatches even on the last two bases. See [http://pubmed.gov/9973624 Mizuno et al., 1999] for example of priming over mismatches. |
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We use the in-house command <code>PipelinePairedEndExtraction.pl</code>. It generates pairs of FASTQ files (5′ and 3′). |
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=== 3′ === |
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| ⚫ | The 6 first bases of the 3′ reads are trimmed because they derive from to the random part (N6) of the reverse-transcription primer, and therefore may not reflect the RNA sequences accurately, since the reverse-transcriptase tolerates mismatches even on the last two bases. See [http://pubmed.gov/9973624 Mizuno et al., 1999] for example of priming over mismatches. |
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=== Output === |
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Pairs of FASTQ files (5′ and 3′), where all the reads originate from the same RNA sample and all the linkers have been trimmed. |
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== Final Output == |
== Final Output == |
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Revision as of 18:10, 4 April 2011
Sample splitting and linker removal
Input is 5′ and 3′ paired-end fastq files from the Illumina sequencers.
- The 9 first bases of the 5′ reads are trimmed. The 6 first are the index sequence (“barcode”) and the 3 next are the linker (
GGG).
- The 6 first bases of the 3′ reads are trimmed because they derive from to the random part (N6) of the reverse-transcription primer, and therefore may not reflect the RNA sequences accurately, since the reverse-transcriptase tolerates mismatches even on the last two bases. See Mizuno et al., 1999 for example of priming over mismatches.
We use the in-house command PipelinePairedEndExtraction.pl. It generates pairs of FASTQ files (5′ and 3′).
Final Output
Mapped paired-end tags in BAM format
