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   "source": [
    "# Working with Paired-Reads\n",
    "If we had paired-end read data, we would need to do things a little bit different at some of the steps.\n"
   ]
  },
  {
   "cell_type": "markdown",
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   "source": [
    "## Running fastq-mcf on Paired Data\n",
    "It only takes two minor changes to run fastq-mcf on paired data, we need to tell it to also load the read 2 file, and also what to call the trimmed output from this file. \n",
    "\n",
    "1. neb_e7600_adapters.fasta\n",
    "2. 27_MA_P_S38_L002_R1_001.fastq.gz\n",
    "2. 27_MA_P_S38_L002_R2_001.fastq.gz : **NEW for paired-data**\n",
    "3. -q 20\n",
    "4. -x 0.5\n",
    "5. -o 27_MA_P_S38_L002_R1_001.trim.fastq.gz\n",
    "6. -o 27_MA_P_S38_L002_R2_001.trim.fastq.gz : **NEW for paired-data**\n",
    "\n",
    "Like this:\n",
    "\n",
    "```\n",
    "fastq-mcf $MYINFO/neb_e7600_adapters.fasta \\\n",
    "    $RAW_FASTQS/27_MA_P_S38_L002_R1_001.fastq.gz \\\n",
    "    $RAW_FASTQS/27_MA_P_S38_L002_R2_001.fastq.gz \\\n",
    "    -q 20 -x 0.5 \\\n",
    "    -o $TRIMMED/27_MA_P_S38_L002_R1_001.trim.fastq.gz \\\n",
    "    -o $TRIMMED/27_MA_P_S38_L002_R2_001.trim.fastq.gz\n",
    "```\n",
    "\n",
    "> Note: Now that, since we are now including the reverse reads, we expect to see contamination with both adapters now"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Running STAR on Paired Data\n",
    "As with fastq-mcf, running STAR on Paired Data on requires a minor change: adding the R2 FASTQ file to the arguments for `--readFilesIn` and removing the \"R1\" from the `--outFileNamePrefix`, since the output will combine R1 and R2, like this:\n",
    "\n",
    "```\n",
    "STAR \\\n",
    "    --runMode alignReads \\\n",
    "    --twopassMode None \\\n",
    "    --genomeDir $GENOME_DIR \\\n",
    "    --readFilesIn $TRIMMED/27_MA_P_S38_L002_R1_001.trim.fastq.gz \\\n",
    "                  $TRIMMED/27_MA_P_S38_L002_R2_001.trim.fastq.gz \\\n",
    "    --readFilesCommand gunzip -c \\\n",
    "    --outFileNamePrefix ${STAR_OUT}/27_MA_P_S38_L002_ \\\n",
    "    --quantMode GeneCounts \\\n",
    "    --outSAMtype BAM Unsorted \\\n",
    "    --outSAMunmapped Within\n",
    "```"
   ]
  }
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