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Overview

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After raw sequence files are generated (in FASTQ format), quality-checked, and pre-processed in some way, the next step in most NGS pipelines is mapping to a reference genome. For individual sequences, it is common to use a tool like BLAST to identify genes or species of origin. However, a normal NGS dataset will have tens to hundreds of millions of sequences, which BLAST and similar tools are not designed to handle.

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titleAnswer
There are 1,184,360 alignment records.

Exercise: How many sequences were in the R1 and R2 FASTQ files combined?

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gunzip -c fastq/Sample_Yeast_L005_R1.cat.fastq.gz | echo $((`wc -l` / 2))

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titleAnswer
There were a total of 1,184,360 original sequences

Exercises:

  • Do both R1 and R2 reads have separate alignment records?
  • Does the SAM file contain both aligned and un-aligned reads?
  • What is the order of the alignment records in this SAM file?
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titleAnswers
  • Do both R1 and R2 reads have separate alignment records?
    • yes, they must, because there were 1,184,360 R1+R2 reads and an equal number of alignment records
  • Does the SAM file contain both aligned and un-aligned reads?
    • yes, it must, because there were 1,184,360 R1+R2 reads and an equal number of alignment records
  • What is the order of the alignment records in this SAM file?
    • the names occur in the exact same order as they did in the FASTQ, except that they come in pairs
      • the R1 read comes first, then its corresponding R2
    • this ordering is called read name ordering

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