Commit eb77c3ad authored by TomKellyGenetics's avatar TomKellyGenetics
Browse files

support Smart-Seq3 with dedicated subroutine

parent b920a666

README.Rmd

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@@ -155,7 +155,7 @@ default settings, see the [installation](#Uninstalling) or [troubleshooting](#De 
-  Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536

-  SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq

-  SeqWell (12bp barcode, 8bp UMI): seqwell

-  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad

-  Smart-seq2-UMI, Smart-seq3 (16bp barcode, 8bp UMI): smartseq



All technologies support 3' single-cell RNA-Seq. Barcode adjustments and

whitelists are changed automatically. For 5' single-cell RNA-Seq, this
@@ -167,7 +167,9 @@ We are developing technologies to support dual indexes and full length scRNA kit 
Experimental technologies (not yet supported):

-  inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3

-  Sci-Seq (8bp UMI, 10bp barcode): sciseq

-  Smart-seq2-UMI, Smart-seq3 (11bp barcode, 8bp UMI): smartseq

-  SPLiT-Seq (10bp UMI, 18bp barcode): splitseq

-  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad





#### Dual-indexing

README.html

+2 −2
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@@ -61,11 +61,11 @@ 
<li>Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536</li>

<li>SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq</li>

<li>SeqWell (12bp barcode, 8bp UMI): seqwell</li>

<li>SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad</li>

<li>Smart-seq2-UMI, Smart-seq3 (16bp barcode, 8bp UMI): smartseq</li>

</ul>

<p>All technologies support 3' single-cell RNA-Seq. Barcode adjustments and whitelists are changed automatically. For 5' single-cell RNA-Seq, this is only supported for 10x Genomics version 2 chemistry. This is detected automatically but can be configured with the <code>--chemistry</code> argument.</p>

<p>We are developing technologies to support dual indexes and full length scRNA kits.</p>

<p>Experimental technologies (not yet supported): - inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3 - Sci-Seq (8bp UMI, 10bp barcode): sciseq - Smart-seq2-UMI, Smart-seq3 (11bp barcode, 8bp UMI): smartseq</p>

<p>Experimental technologies (not yet supported): - inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3 - Sci-Seq (8bp UMI, 10bp barcode): sciseq - SPLiT-Seq (10bp UMI, 18bp barcode): splitseq - SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad</p>

<h4 id="dual-indexing">Dual-indexing</h4>

<p>For dual-indexed technologies such as inDrops-v3, Sci-Seq, SmartSeq3 it is advised to use &quot;bcl2fastq&quot; before calling UniverSC:</p>

<pre><code>   /usr/local/bin/bcl2fastq  -v --runfolder-dir &quot;/path/to/illumina/bcls&quot;  --output-dir &quot;./Data/Intensities/BaseCalls&quot;\

README.md

+4 −2
Original line number Diff line number Diff line
@@ -155,7 +155,7 @@ default settings, see the [installation](#Uninstalling) or [troubleshooting](#De 
-  Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536

-  SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq

-  SeqWell (12bp barcode, 8bp UMI): seqwell

-  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad

-  Smart-seq2-UMI, Smart-seq3 (16bp barcode, 8bp UMI): smartseq



All technologies support 3' single-cell RNA-Seq. Barcode adjustments and

whitelists are changed automatically. For 5' single-cell RNA-Seq, this
@@ -167,7 +167,9 @@ We are developing technologies to support dual indexes and full length scRNA kit 
Experimental technologies (not yet supported):

-  inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3

-  Sci-Seq (8bp UMI, 10bp barcode): sciseq

-  Smart-seq2-UMI, Smart-seq3 (11bp barcode, 8bp UMI): smartseq

-  SPLiT-Seq (10bp UMI, 18bp barcode): splitseq

-  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad





#### Dual-indexing

launch_universc.sh

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Original line number Diff line number Diff line
@@ -209,6 +209,7 @@ Mandatory arguments to long options are mandatory for short options too. 
                                  Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536

                                  SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq

                                  SeqWell (12bp barcode, 8bp UMI): seqwell

                                  Smart-seq2-UMI, Smart-seq3 (16bp barcode, 8bp UMI): smartseq

                                  SPLiT-Seq (10bp UMI, 18bp barcode): splitseq

                                  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad

                                Custom inputs are also supported by giving the name "custom" and length of barcode and UMI separated by "_"
@@ -217,7 +218,6 @@ Mandatory arguments to long options are mandatory for short options too. 
                                Experimental technologies (not yet supported):

                                  inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3

                                  Sci-Seq (8bp UMI, 10bp barcode): sciseq                                  

                                  Smart-seq2-UMI, Smart-seq3 (11bp barcode, 8bp UMI): smartseq



  -b,  --barcodefile FILE       Custom barcode list in plain text (with each line containing a barcode)
@@ -650,6 +650,8 @@ if [[ "$technology" == "icell8" ]]; then 
fi

if [[ "$technology" == "smartseq" ]]; then

    echo "***WARNING: ${technology} should only be used for kits that have UMIs***"

    echo "... UMI reads will be filtered using a tag sequence which will be removed""

    echo "... barcodes will derived from dual indexes"

fi

if [[ "$technology" == "smartseq" ]] || [[ "$technology" == "indrop-v1" ]] || [[ "$technology" == "indrop-v2" ]] || [[ "$technology" == "indrop-v3" ]]; then

    echo "***WARNING: launch_universc.sh does not support barcodes in dual indexes. Make sure that the R1 file is adjusted accordingly prior to running launch_universc.sh***"
@@ -735,9 +737,9 @@ elif [[ "$technology" == "seqwell" ]]; then 
    umilength=12

    minlength=8

elif [[ "$technology" == "smartseq" ]]; then

    barcodelength=11

    barcodelength=16

    umilength=8

    minlength=11

    minlength=16

elif [[ "$technology" == "splitseq" ]]; then

    barcodelength=18

    umilength=10
@@ -2291,8 +2293,10 @@ else 
            convI1=$(echo $read | perl -pne 's/(.*)_R1/$1_I1/' )

            convI2=$(echo $read | perl -pne 's/(.*)_R1/$1_I2/' )



            echo "  ...remove internal for ${technology} by matching tag sequence for UMI reads"

            # filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters 

            perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1} --r2=${convR2} --i1=${convI1} --i2=${convI2} --out_dir $crIN

            echo "  ...trim tag sequence from R1"



            # returns R1 with tag sequence removed (left trim) starting with 8pbp UMI and corresponding reads for I1, I2, and R2

            mv $crIN/SmartSeq3_parsed_R1.fastq ${convR1}
@@ -2300,6 +2304,7 @@ else 
            mv $crIN/SmartSeq3_parsed_I1.fastq ${convI1}

            mv $crIN/SmartSeq3_parsed_I2.fastq ${convI2}



            echo "  ...concatencate barcodes to R1 from I1 and I2 index files"

            # filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters 

            perl sub/ConcatenateDualIndexBarcodes.pl --additive=${convI1} --additive=${convI2} --ref_fastq=${convR1} --out_dir $crIN

man/launch_universc.sh

+1 −1
Original line number Diff line number Diff line
@@ -199,6 +199,7 @@ Provides a conversion script to run multiple technologies and custom libraries w 
                                  Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536

                                  SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq

                                  SeqWell (12bp barcode, 8bp UMI): seqwell

                                  Smart-seq2-UMI, Smart-seq3 (16bp barcode, 8bp UMI): smartseq

                                  SPLiT-Seq (10bp UMI, 18bp barcode): splitseq

                                  SureCell (18bp barcode, 8bp UMI): surecell, ddseq, biorad

                                Custom inputs are also supported by giving the name "custom" and length of barcode and UMI separated by "_"
@@ -207,7 +208,6 @@ Provides a conversion script to run multiple technologies and custom libraries w 
                                Experimental technologies (not yet supported):

                                  inDrops version 3 (8bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3

                                  Sci-Seq (8bp UMI, 10bp barcode): sciseq                                  

                                  Smart-seq2-UMI, Smart-seq3 (11bp barcode, 8bp UMI): smartseq



           A barcode whitelist is provided for all beads or wells for the following technologies:

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